• IMMUNE NETWORK
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• v.13 no.1
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• pp.16-24
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• 2013

CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1613-1619
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• 2016

Francisella tularensis is a highly virulent pathogen of humans and other mammals. Moreover, F. tularensis has been designated a category A biothreat agent, and there is growing interest in the development of a protective vaccine. In the present study, we determine the in vitro and in vivo immune responses of a subunit vaccine composed of recombinant peptides Tul4 and FopA from epitopes of the F. tularensis outer membrane proteins. The recombinant peptides with adjuvant CpG induced robust immunophenotypic change of dendritic cell (DC) maturation and secretion of inflammatory cytokines (IL-6, IL-12). In addition, the matured DCs enabled ex vivo proliferation of naive splenocytes in a mixed lymphocyte reaction. Lastly, we determined the in vivo immune response by assessment of antibody production in C57BL/6 mice. Total IgG levels were produced after immunization and peaked in 6 weeks, and moreover, Tul4-specific IgG was confirmed in the mice receiving peptides with or without CpG. Based on these results, we concluded that the recombinant peptides Tul4 and FopA have immunogenicity and could be a safe subunit vaccine candidate approach against F. tularensis.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.82-82
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• 1995

4-lBB molecule is expressed on the surface of activated CD4$\^$+/ and CD8$\^$+/ T cells. We generated a panel of anti-4-1 B5 murine mAbs using a fusion protein consisting of the extracellular domain of human 4-1 BB fused to Glutathione S-transferase. The binding activity against cell surface 4-1 BB molecule was assessed by flow cytometry analysis. These studies showed that several anti-4-1 BB mAbs bound to 10-30% of CD4$\^$+/ and CD8$\^$+/T cells in PHA or Con A stimulated PBLs, although these mAbs interacted with only, l-2% of CD4$\^$+/ and CD8$\^$+/ T cells in normal PBLs, indicating the specificity of mAbs to the 4-l BB molecule on activated CD4$\^$+/ and CD8$\^$+/ T cells. Next, we examined the effect of an anti-4-l BB mAb (4B4-1-1) on allogeneic mixed lymphocyte reactions (MLRs). The data indicated that the antibody significantly inhibited the proliferative response at higher concentrations. When tested with several T cell mitogens, the antibody had no stimulatory or inhibitory effects on the mitogen-mediated T cell proliferation. These data suggest that 4-1 BB molecule may play a role in the regulation of antigen-mediated immune response.

• The Journal of the Korean Society for Microbiology
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• v.18 no.1
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• pp.59-65
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• 1983

To investigate the effects of placental serum to in vitro culture of the normal human lymphocytes the peripheral blood lymphocytes were isolated by ficoll-hypaque separation method in 20 healthy human adults. The blast transformation respone of lymphocytes to mitogens were observed by stimulation with PHA($25{\mu}g/ml$) and Con A($25{\mu}g/ml$) using RPMI 1640 media containing 20% placental serum(PS), tetal calf serum(FCS), or humans AB serum(AB). And one-way mixed lymphocyte culture test was performed between these unrelated person compounded into stimulators and responders to investigate the effect of placental serum. The following results were obtained. 1. In 20 experimental cases, these were no significant diffenence between FCS, AB, and PS in untreated control cultures. 2. In PHA-treated cultures, whereas the blast transformation rate of the FCS groups and AB groups were $40.8{\pm}4.3%$ and $44.6{\pm}4.3%$ respectively, that of PS groups were $21.7{\pm}3.4%$, Similar results were obtained in Con A-meated cultures. Therefore, placental serum inhibited the mitogenic response of lymphocytes significantly. 3. In MLC tests, stimulation index of the FCS groups and AB groups were $18.5{\pm}3.5$ and $20.1{\pm}3.3$ respectively. But placental serum inhibited MLC response of lymphocytes significantly($7.4{\pm}1.9$).

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.10
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• pp.1416-1424
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• 2017

Objective: The objective of this study was to examine the effects of alfalfa flavonoids on the production performance, immunity, and ruminal fermentation of dairy cows. Methods: The experiments employed four primiparous Holstein cows fitted with ruminal cannulas, and used a $4{\times}4$ Latin square design. Cattle were fed total mixed ration supplemented with 0 (control group, Con), 20, 60, or 100 mg of alfalfa flavonoids extract (AFE) per kg of dairy cow body weight (BW). Results: The feed intake of the group receiving 60 mg/kg BW of AFE were significantly higher (p<0.05) than that of the group receiving 100 mg/kg BW. Milk yields and the fat, protein and lactose of milk were unaffected by AFE, while the total solids content of milk reduced (p = 0.05) linearly as AFE supplementation was increased. The somatic cell count of milk in group receiving 60 mg/kg BW of AFE was significantly lower (p<0.05) than that of the control group. Apparent total-tract digestibility of neutral detergent fiber and crude protein showed a tendency to increase (0.05<$p{\leq}0.10$) with ingestion of AFE. Methane dicarboxylic aldehyde concentration decreased (p = 0.03) linearly, whereas superoxide dismutase activity showed a tendency to increase (p = 0.10) quadratically, with increasing levels of AFE supplementation. The lymphocyte count and the proportion of lymphocytes decreased (p = 0.03) linearly, whereas the proportion of neutrophil granulocytes increased (p = 0.01) linearly with increasing levels of dietary AFE supplementation. The valeric acid/total volatile fatty acid (TVFA) ratio was increased (p = 0.01) linearly with increasing of the level of AFE supplementation, the other ruminal fermentation parameters were not affected by AFE supplementation. Relative levels of the rumen microbe Ruminococcus flavefaciens tended to decrease (p = 0.09) quadratically, whereas those of Butyrivibrio fibrisolvens showed a tendency to increase (p = 0.07) quadratically in response to AFE supplementation. Conclusion: The results of this study demonstrate that AFE supplementation can alter composition of milk, and may also have an increase tendency of nutrient digestion by regulating populations of microbes in the rumen, improve antioxidant properties by increasing antioxidant enzyme activities, and affect immunity by altering the proportions of lymphocyte and neutrophil granulocytes in dairy cows. The addition of 60 mg/kg BW of AFE to the diet of dairy cows was shown to be beneficial in this study.

• Archives of Plastic Surgery
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• v.32 no.4
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• pp.533-538
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• 2005

Due to its safety and softness, autologous fat transplantation has been commonly performed for soft tissue correction. However, the injected fat is absorbed resulting in the reduction of volume of the graft by 40-60% within a few months. Thus, there was an attempt to use adipocytes differentiated from preadipocytes in vitro for transplantation. Differentiated adipocytes were biocompatible and matured with gradual volume increase at transplantation site in clinical study(unpublished data). In addition, they did not induce immune rejection in response to nonself lymphocytes in a mixed lymphocyte reaction(MLR)(unpublished data). The purpose of this study is to differentiate mouse preadipocytes following labeling into adipocytes to establish an animal model for allogenic transplantation. Preadipocytes isolated from inguinal and retroperitoneal fat pad of C57BL/6 mice were proliferated with growth medium by passage 3 and differentiated into adipocytes with different culture conditions after labeled with BrdU. At most suitable conditions, above 90% of preadipocytes were differentiated and BrdU labeling did not affect differentiation rate and function of differentiated adipocytes. These results demonstrate that BrdU-labeled adipocytes resulting from this in vitro differentiation protocol are useful for allogenic transplantation study.

• The Journal of Internal Korean Medicine
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• v.25 no.4
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• pp.117-128
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• 2004

Objectives : To examine the effects of Gojineumja on white rats which deteriorated immunity caused by Methotrexate(MTX), first of all, MTX was fed to the rats once a day for 4 day. Methods : After the immune response of the rats are deteriorated, dried extracts of Gojineumja(GJE) mixed in water was fed to the white rats once a day for l4days. The next conclusion was made by examining the rates of B-cells and T-cells of the peripheral blood and the changes in rates of CD4+ T-cells and CD8+ T -cells of the blood sampled from the spleen and peripheral region. Especially the count of CD3+ CD4+ T-cells of the peripheral blood and the count of CD3+ CD4+ T-cells of the spleen the count of CD4+/ CD8+ T-cell of the peripheral blood and the spleen proved the significant effect of increasing immune responses statistically. Results :(1) The following are the summary of the results. (2) The percentage of B lymphocyte of peripheral blood was increased significantly in GJE group as compared with control group. (3) The percentage of CD3+ CD4+ T-cell of peripheral blood was increased significantly in GJE group as compared with control group. (4) The percentage of CD3+ CD8+ T-cell of peripheral blood was not different statistically. (5) The percentage of CD4+/ CD8+ T-cell of peripheral blood was increased significantly in GJE group as compared with control group. (6) The percentage of CD3+ CD4+ T-cell of spleen was increased significantly in GJE group as compared with control group. (7) The percentage of CD3+ CD8+ T-cell was not different statistically. (8) The percentage of CD4+ /CD8+ T-cell was not different statistically. Conclusions : Gojineumja has an effect of increasing immune responses on white rats with deteriorated immunity caused by MTX.

• IMMUNE NETWORK
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• v.5 no.4
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• pp.221-231
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• 2005

Background: Immune regulatory dendritic cells (DCs) play an important role in maintaining self-tolerance. Recent evidences demonstrate that DCs expressing indoleamine 2,3-dioxygenase (IDO), which is involved in tryptophan catabolism, play an important role in immunoregulation and tolerance and induce T cell apoptosis. This study was devised to examine the role of IDO in the oral tolerance induction in collagen-induced arthritis (CIA) mouse model. Methods: Beginning 2 weeks before immunization, CII was fed six times to DBA/1 mice and the effect on arthritis was assessed. In tolerized mice, $CD11c^+$ DCs were isolated and stimulated with CII, IFN-${\gamma}$, and LPS with or without IDO inhibitor, 1-methyl-DL-tryptophan (1-MT) and IDO expression by $CD11c^+$ DCs was analyzed using FACS and RT-PCR. The expression of IDO, MHC II, CD80, and CD86 by $CD11c^+$ DCs were examined using confocal microscopy. Regulatory effect of $CD11c^+$ DCs on Ag-specific T cell proliferative response to CII was examined by mixed lymphocyte reaction (MLR) with or without 1-MT. Results: The proportion of IDO-expressing $CD11c^+$ DCs was slightly higher in tolerized mice than in CIA mice and significantly increased after stimulation with CII, IFN-${\gamma}$, and LPS in an IDO-dependent manner. On confocal microscopic examination, the expression of IDO was higher and those of MHC II and CD86 were lower in CD11c + DCs from tolerized mice compared to those from CIA mice. On MLR, $CD11c^+$ DCs from tolerized mice inhibited T cell proliferative response to CII in an IDO-dependent manner. Conclusion: Enhanced IDO expression by $CD11c^+$ DCs from tolerized mice may contribute to the regulation of proliferative response of CII-reactive T cells and could be involved in the induction of oral tolerance to CII.

• IMMUNE NETWORK
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• v.11 no.1
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• pp.79-94
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• 2011

Background: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-${\kappa}B$. Methods: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma. Results: DPT promoted the activation of $CD8^+$ T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-${\gamma}$ production and induction of cytotoxic T lymphocyte activity. Conclusion: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.

• Archives of Plastic Surgery
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• v.35 no.4
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• pp.385-392
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• 2008

Purpose: The most effective methods of harvesting, preparing, and injecting autologous fat grafts have been inconsistent and conflicting. With its limitation as resorption in fat grafting, handling various techniques affect adipocyte survival, and is crucial to optimizing its long-term survival. To improve graft survival, re-implantation of cryopreserved adipocytes was developed. In addition, adipocytes do not induce immune rejection in response to non-self lymphocytes in a mixed lymphocyte reaction. The purpose of this study is to analyze the changes in cryopreserved adipocytes so as to determine the most efficient long-term storage period, and to analyze the changes in cryopreserved allografted adipocytes so as to determine the efficacy of cryopreserved adipocytes allografting. Methods: Fat tissues were harvested from the inguinal and retroperitoneal fat pad of mice. After the centrifugation of the harvested fat tissues, they were disintegrated with collagenase. The adipocytes were obtained by centrifugation of the disintegrated fat tissues. The adipocytes were treated as follows: (1) They were examined for weight and then frozen at $-20^{\circ}C$(n=25). For four months, each five frozen samples were taken and examined for weight and histologic changes in the 1st week, the 1st month, the 2nd month, the 3rd month, and the 4th month, respectively. (2) The adipocytes were immediately frozen at $-20^{\circ}C$(n=125). For four months, five frozen samples were taken, and allografted in the same time period as above. Finally, for four months, five cryopreserved allografted adipocytes were taken and examined for histologic changes in the same time period as above. Results: (1) Significant weight changes and histologic findings with inflammatory and destructive changes were observed in the cryopreserved adipocytes in three months. (2) Significant fat necrotic changes in the histologic changes with Hematoxylin and eosin stain were observed in the cryopreserved allografted adipocytes since the first week, independent of the freezing period. Conclusion: The study results show that the adipocytes that were cryopreserved for more than three months underwent obvious weight reductions and necrotic changes, and the adipocytes that were allografted without freezing were viable for four months, but the cryopreserved allografted adipocytes had obvious necrotic changes since the first week regardless of the freezing period.