• IMMUNE NETWORK
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• v.8 no.1
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• pp.21-28
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• 2008

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.

• Korean Journal of Food Science and Technology
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• v.40 no.5
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• pp.574-579
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• 2008

Propolis is the generic term for the resinous substance collected by honey bees from a variety of plant sources. In this study, we have assessed the immunomodulatory properties of propolis (P) and fermented-propolis (FP) in BALB/c mice. Mice were subjected to gavage once a day (for 14 days) with 50, 100, 200 mg/kg body weight P, FP, or vehicle. Lymphocytes were isolated from the spleen and mesenteric lymph nodes (MLN) and the immune cell proportions, proliferative activities, and cytokine production were evaluated. The P- and FP-administration induced similar, but differential, alterations in the percentage of immune cell populations and their biological functions, including cytokine production and NK cell cytotoxicity. The proportion of$ CD4^+$ and $CD8^+$ T cells in the spleen was increased slightly in the P- and FP-administered mice as compared to the vehicle-treated mice. In MLN, the percentage of $CD4^+$ T cells was increased significantly in the 200 mg/kg P-treated mice. The mice which were treated with P and FP evidenced significantly increased interferon-$\gamma$ and interleukin-4 production in concanavalin A-stimulated splenocytes, whereas the production of theses cytokines was not shown to be induced by P-treatment. In addition, NK cell activity was also increased dramatically by the administration of P and FP. Collectively, these findings showed that P and FP are wide-spectrum immunomodulators, which may modulate both innate and adaptive immune responses.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1326-1334
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• 2008

The prime-boost vaccination with DNA vaccine and recombinant viral vector has emerged as an effective prophylactic strategy to control infectious diseases. Here, we compared the protective immunities induced by multiple alternating immunizations with DNA vaccine (pCIgB) and replication-incompetent adenovirus (Ad-gB) expressing glycoprotein gB of pseudorabies virus (PrV). The platform of pCIgB-prime and Ad-gB-boost induced the most effective immune responses and provided protection against virulent PrV infection. However, priming with pCIgB prior to vaccinating animals by the DNA vaccine-prime and Ad-boost protocol provided neither effective immune responses nor protection against PrV. Similarly, boosting with Ad-gB following immunization with DNA vaccine-prime and Ad-boost showed no significant responses. Moreover, whereas the administration of Ad-gB for primary immunization induced Th2-type-biased immunity, priming with pCIgB induced Th1-type-biased immunity, as judged by the production of PrV-specific IgG isotypes and cytokine IFN-$\gamma$. These results indicate that the order and injection frequency of vaccine vehicles used for heterologous prime-boost vaccination affect the magnitude and nature of the immunity. Therefore, our demonstration implies that the prime-boost protocol should be carefully considered and selected to induce the desired immune responses.

• The Journal of Pediatrics of Korean Medicine
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• v.25 no.1
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• pp.1-27
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• 2011

Objectives: The purpose of this study is to investigate the effects of Yijungtang(YJT) on atopic dermatitis in an in-vitro and in-vivo experiment using a RBL-2H3 mast cells and a NC/Nga atopic dermatitis mouse. Methods: In-vitro experiment, IL-4, IL-13 mRNA expression were evaluated by a real-time PCR, IL-4, IL-13 production by ELISA and transcription factor as GATA-1, GATA-2, NF-AT1, NF-AT2, AP-1 and NF-kB by western blotting. In-vivo experiment, clinical skin score we evaluated by, hematology and Serum total IgE and IgG1 of NC/Nga atopic dermatitis mouse, cytokine level, total number of cell, Immunohistochemical staining and Histological features of auxiliary lymph node(ALN), draining lymph node(DLN), peripheral blood mononuclear cells(PBMCs) and dorsal skin tissue in NC/Nga mouse. Results: YJT decreased IL-4, IL-13 mRNA expression, IL-4, IL-13 production and prominently decreased the expression of mast cell specific transcription factors including GATA-2, NF-AT2, c-Fos and NF-kB. YJT oral administration reduced the levels of skin severity scores. It also decreased the level of inflammatory cytokines such as IL-5, IL-13, histamine and IgE in the serum. It elevated IFN-gamma level in the spleenocyte culture supernatant but decreased. $CD3e^+$, $CD19^+$, $CD4^+$, $CD8^+$, $CD3e^+CD69^+$, $CD11b^+Gr-1^+$, $CCR3^+$ in the PBMCs, $CD4^+$, $CD8^+$, $CD3e^+CD69^+$, $B220^+CD23^+$ in the ALN, $CD4^+$, $CD3e^+CD69^+$ in the ALN and $CD4^+$, $CD11b^+Gr-1^+$ in the dorsal skin. Histological examination showed that infiltration levels of immune cells in the skin of AD-induced NC/Nga mice were much improved by YJT oral administration. Conclusions: The anti-allergic activities of YJT may be mediated by down-regulation of Th2 cytokines, such as IL-4 and IL-13, through the regulation GATA-2, NF-AT2 and NF-kB transcription factors in mast cells. YJT would be regulate molecular mediators and immune cells which are functionally associated with atopic dermatitis induced in NC/Nga mice, and may play an important role in recovering AD symptoms.

• IMMUNE NETWORK
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• v.3 no.2
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• pp.136-144
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• 2003

Oral administration of antigen has long been considered as a promising alternative for the treatment of chronic autoimmune diseases including rheumatoid arthritis (RA), and oral application of type II collagen (CII) has been proven to improve pathogenic symptoms in RA patients without problematic side effects. To further current understandings about the immune suppression mechanisms mediated by orally administered antigens, we examined the changes in IgG subtypes, T-cell proliferative response, and proportion of interleukin (IL)-10 producing Th subsets in a time course study of collagen induced arthritis (CIA) animal models. We found that joint inflammation in CIA mouse peaked at 5 weeks after first immunization with CII, which was significantly subdued in mice pre-treated by repeated oral administration of CII. Orally tolerized mice also showed increase in their serum level of IgG1, while the level of IgG2a was decreased. T-cell proliferation upon CII stimulation was also suppressed in lymph nodes of mice given oral administration of CII compared to non-tolerized controls. When cultured in vitro in the presence of CII, T-cells isolated from orally tolerized mice presented higher proportion of $CD4^+IL-10^+$ subsets compared to non-tolerized controls. Interestingly, such increase in IL-10 producing cells were obvious first in Peyer's patch, then by 5 weeks after immunization, in mesenteric lymph node and spleen instead. This result indicates that a particular subset of T-cells with immune suppressive functions might have migrated from the original contact site with CII to inflamed joints via peripheral blood after 5 weeks post immunization.

• Journal of Periodontal and Implant Science
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• v.29 no.1
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• pp.1-14
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• 1999

Ursodeoxycholic acid(UDCA) is a hydrophilic gall bladder acid and has been used as a effective drug for liver disease related to in1munity. This drug inhibits secretions of IL-2, IL-4, and $IFN-{\gamma}$ from T-cells and production of immunoglobulin from B-cells. Also it has been reported that UDCA inhibits production of IL-1 related to the progression of periodontal disease and activation of collagenases. The purpose of the present study was to elucidate the effects of UDCA on inhibition of periodontal disease progression using clinical, microbiological and histometrical parameters. Twelve pure bred, 16 month-old-beagle dogs were used in the study. After ligature-induced periodontal diseases were formed, experimental drugs were applied twice a day and then the results of clinical, microbiological, and histometrical parameters were measured at baselie(initiation of experiment) , 4weeks and 8weeks. The gel with UDCA(concentration 0.5%, 5% 3 dogs in each) was applied to experimental group, chlorhexidine to positive control group(3dogs) and the gel without UDCA(base) to negative control group. After induction of general anesthesia, the maxillary 2nd, 3rd premolars and 1st molar and the mandibular 2nd, 3rd, 4th premolars and 1st molar were ligated in one side selected randomly and were not ligated in the opposite side. The plaque index(PI), gingival index(GI), pocket depth(PD) and gingival crevicular fluid(GCF) volum were measured clinically. The PI and GI were measured at 3 buccal points of all experimental teeth and the GCF was measured only at the 3rd premolar in the maxilla and the 4th premolar in the mandible. In the microbiological study, the samples extracted from the 3rd premolar of the maxilla and the 4th premolar of the mandible at the center of buccal surface were analyzed aerobics, anaerobics and Streptococcus colony forming units, After clinical and microbiological examination at 8weeks, the dogs were sacrificed by carotid artery perfusion. The samples were fixed and sectioned including interproximal area, and the distance from cementoenamel junction(CEJ) to alveolar crest was measured. The results were that PI, GI and PD increased until 4 weeks and decreased at 8 weeks in three groups but the differences between all the groups were not significant. The 0.5% UDCA in non-ligated group showed remarkable decrease of GCF. The experimental group applied 5% UDCA decreased the number of aerobics and anaerobics. The distance from CEJ to alveolar crest was greater in the negative control group on both ligated and non-ligated sides, but the differences were not significant stastically.

• IMMUNE NETWORK
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• v.6 no.3
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• pp.154-162
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• 2006

Background: Dendritic cell (DC)-based cancer immunotherapy is studied for several years. However, it is mainly derived from autologous PBMC or leukapheresis from patient, which has limitations about yield and ability of DC production according to individual status. In order to solve these problems, inquiries about allogeneic DCs are performed but there are no preclinical trial answers for effect or toxicity of allogeneic DC to use for clinical trial. In this study, we compared the anti-tumor effect of allogeneic and autologous DCs from mouse bone marrow stem cells in mouse metastatic melanoma model. Methods: B16F10 melanoma cells ($5{\times}10^4$/mouse) were injected intravenously into the C57BL/6 mouse. Therapeutic DCs were differentiated from autologous (C57BL/6: CDC) or allogeneic (B6C3F1: BDC) bone marrow stem cells with GM-CSF, SCF and IL-4 for 13days and pulsed with B16F10 tumor cell lysate (Blys) for 18hrs. DC intra-peritoneal injections began on the 8th day after the tumor cell injection by twice with one week interval. Results: Anti-tumor response was observed by DC treatment without any toxicity especially in allogeneic DC treated mice (tumor burden score: $2.667{\pm}0.184,\;2.500{\pm}0.463,\;2.000{\pm}0.286,\;1.500{\pm}0.286,\;1.667 {\pm}0.297$ for saline, CDC/unpulsed-DC: U-DC, CDC/Blys-DC, BDC/U-DC and BDC/Blys-DC, respectively). IFN-${\gamma}$ secretion was significantly increased in allogeneic DC group stimulated with B16F10 cell lysate ($2,643.3{\pm}5,89.7,\;8,561.5{\pm}2,204.9.\;6,901.2{\pm}141.1pg/1{\times}10^6$ cells for saline, BDC/U-DC and BDC/Blys-DC, respectively) with increased NK cell activity. Conclusion: Conclusively, promising data was obtained that allogeneic DC can be used for DC-based cancer immunotherapy.

• The Journal of Pediatrics of Korean Medicine
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• v.22 no.1
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• pp.69-93
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• 2008

Objectives The purpose of this study is to investigate the effect of KKHS on atopic dermatitis in an in-vivo experiment using an NC/Nga atopic dermatitis mouse, which has histological and clinical similarities to this condition in humans. Methods To investigate the effect of KKHS on atopic dermatitis (AD), we evaluated atopic dermatitis-like skin lesions by clinical skin index and analyzed immunological parameters in peripheral blood mononuclear cells(PBMCs), splenocytes, draining lymph node(DLN) and performed skin histology in ears and dorsal skin of atopic dermatitis of NC/Nga mouse in vivo. Results In vivo, clinical skin severity score was significantly lower in the KKHS group than in the control group. IgE, IL-6, TNF-${\alpha}$, IgM, IgG2a and IgG2b levels in serum decreased remarkably in the KKHS group than in the control group, and the level of IFN-${\gamma}$ production which is secreted from Th1 cell was increased by KKHS. After this experiment we analyzed immunological cells ($CD3^+$, $CD19^+$, $CD4^+$, $CD8^+$, $CD3^+CD69^+$, $CD4^+CD25^+$ and $CD49b^+$) by flow cytometry. It results that the total absolute number of $CD3^+$, $CD19^+$, $CD4^+$ and $CD8^+$ cells were recovered as much as normal state, and the level of $CD3^+CD69^+$ in isolated DLN and PBMCs were significantly decreased, and total absolute number of $Gr-1^+$, $CD11b^+$ and $CD3^+$ in dorsal skin of NC/Nga mouse were decreased by KKHS. We analyzed ear, DLN, and neck-back skin after biopsy and dyeing by hematoxyline/eosin(H&E), toluidine staining (mast cells marker). KKHS were very effective to the histological symptoms which are in dermal and epidermal thickening, hyperkeratosis and inflammatory cell infiltration. Ear thickness was significantly decreased compared with the control group and the size of inflammatory lymphocytes cells (ILC) and plasma cells (PC) in DLN were also decreased. Conclusions KKHS on atopic dermatitis in an in-vivo experiment using an NC/Nga atopic dermatitis mouse was very effectiveness to the atopy dermatitis treatment.

• The Journal of Pediatrics of Korean Medicine
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• v.22 no.2
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• pp.81-114
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• 2008

Objectives : The purpose of this study is to investigate the effect of Jeseupwiryeongtang-gagam(JWTG) on atopic dermatitis by in vivo experiment using NC/Nga atopic dermatitis mouse, which has histological and clinical similarities to the atopic dermatitis of human. Methods : To investigate the effect of JWTG on AD, we evaluated atopic dermatitis-like skin lesions by clinical skin index and analyzed immunological parameters in peripheral blood mononuclear cells(PBMCs), splenocytes, draining lymph node(DLN) and performed skin histology in ears and dorsal skin of atopic dermatitis-like skin NC/Nga mouse in vivo. Results and Conclusions : In vivo, clinical skin severity score were significantly lower in JWTG group than control group. IgE, IL-6, $TNF-{\alpha}$, IgM, IgG2a and IgG2b levels in serum were decreased remarkably in JWTG group than control group and $IFN-{\gamma}$ production, secreted in Th1 cell were increased by JWTG. After experiment ended, we analyzed immunological cells ($CD3^+$, $CD19^+$, $CD4^+$, $CD8^+$, $CD3^+$$CD69^+$, $CD4^+$$CD25^+$ and $CD49b^+$) by flow cytometry. It resulted that total absolute number of $CD3^+$, $CD19^+$, $CD4^+$ and $CD8^+$ cells were recovered as normal and $CD3^+$$CD69^+$ were decreased significantly compared with control group in isolated DLN and PBMCs from NC/Nga mouse and total absolute number of $Gr-1^+$, $CD11b^+$ and $CD3^+$ in dorsal skin of NC/Nga mouse were decreased by JWTG. We analyzed ear, DLN, and neck-back skin after biopsy and dyeing by hematoxyline/eosin(H&E) and toluidine staining (mast cells marker) and obtained results that JWTG were effective to histological symptoms (dermal and epidermal thickening, hyperkeratosis and inflammatory cell infiltration). Ear thickness was decreased significantly than the control group and the size of inflammatory lymphocytes cells(ILC) and plasma cells(PC) in DLN were also decreased.

• Parasites, Hosts and Diseases
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• v.56 no.4
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• pp.325-334
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• 2018

Toxoplasma gondii is an apicomplexan zoonotic protozoan parasite that infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicate a need for the development of an effective vaccine. T. gondii GRA8 is a member of the dense granules protein family and is used as a marker of acute infection. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pDsRed2-GRA8, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with the pDsRed2-GRA8 plasmid and then challenged by infection with the highly virulent GFP-RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii of this vaccine were analyzed by measuring cytokine and serum antibody titers, splenocyte proliferation assays, and the survival times of mice after challenge. Our results showed that mice immunized with pDsRed2-GRA8 demonstrated specific humoral and cellular responses, induced higher IgG antibody titers with predominant IgG2a production; increased levels of IL-10, IL-12 (p70), $IFN-{\gamma}$, $TNF-{\alpha}$, and splenocyte proliferation; and prolonged survival times compared to those of control mice. The present study showed that DNA immunization with pDsRed2-GRA8 induced humoral and cellular immune responses, and all immunized mice showed greater Th1-type immune responses and longer survival times than those of control mice. These results indicated that T. gondii GRA8 DNA immunization induces a partial protective effect against acute toxoplasmosis.