• Journal of Embryo Transfer
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• v.31 no.1
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• pp.1-7
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• 2016

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of $IFN-{\gamma}$. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.166-175
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• 2018

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line ($Rag-2^{-/-}$) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. $Rag-2^{-/-}$ mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in $Rag-2^{-/-}$ mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in $Rag-2^{-/-}$ mice. These findings indicate that our $Rag-2^{-/-}$ mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

• Korean Journal of Food Science and Technology
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• v.51 no.3
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• pp.263-271
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• 2019

In this study, we examined the immunostimulating characteristics of a hot water extract (RCW) and crude polysaccharides (RCP) of red cabbage. RCW and RCP did not show any cytotoxicity in B16BL6 cells and macrophages. Although the sugar compositions of RCW and RCP were similar, the uronic acid content of RCP was higher than that of RCW RCP significantly increased the production of various cytokines and NO, whereas RCW did not affect the production of cytokines and NO. In an ex vivo assay of natural killer (NK) cell activity, intravenous (i.v.) administration of RCP significantly augmented NK cytotoxicity against Yac-1 tumor cells at 3 days after RCP treatment. In an experimental lung metastasis model using B16BL6 melanoma cells, i.v. administration of RCP at a dose of $1,000{\mu}g$ per mouse significantly inhibited 47.3% of lung metastasis. These results suggest that crude polysaccharide isolated from red cabbage is a promising food ingredient for the prevention of tumor metastasis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.2
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• pp.216-223
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• 2014

Curcuma longa L. (CL) is a well known traditional medicinal plant that is also used in curries and mustards as a coloring and flavoring agent. However, CL is not usually used as a food source due to its bitter taste. We investigated the immunomodulatory effect of CL fermented by Aspergillus oryzae (FCL) on RAW 264.7 cells. FCL was extracted with cold water (CW), hot water (HW), 20% ethanol (20% EtOH) and 80% ethanol (80% EtOH), after which its effects on phagocytic activity, tumor necrosis factor-alpha (TNF-${\alpha}$), nitric oxide (NO) production, natural killer (NK) cell activity and mRNA expression of LP-BM5 eco were investigated. Phagocytic activity was increased in HW and 20% EtOH when compared to the control. The secretion of nitric oxide (NO) from RAW 264.7 cells did not change significantly relative to the control. However, TNF-${\alpha}$ was significantly increased by the addition of FCL extracts. Moreover, FCL 20% ethanol extract showed a four fold increase in NK cell cytotoxity relative to the control group. Finally, we observed suppressed mRNA expression of LP-BM5 eco in FCL extracts, especially in the 20% ethanol extracts group. These results indicate that the FCL extracts can be used as a functional material due to their effective immunomodulating activities.

• Korean Journal of Food Science and Technology
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• v.48 no.3
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• pp.268-274
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• 2016

The purpose of this study was to investigate the immuno-stimulatory activity of the high-molecular-weight fraction (HMWF) of Cynanchum wilfordii (CW) extracts obtained by ultrafiltration in murine macrophage RAW 264.7 cells and to assess its immuno-stimulatory effect in mice. Ultrafiltration was performed with polyethersulfone membranes (30 kDa cutoff) in a cross-flow filtration system to obtain the HMWF of CW. The results showed that the HMWF increased the production of various cytokines such as tumor necrosis factor-${\alpha}$, interleukin-6, and nitric oxide in dose-ependent manners. In addition, HMWF treatment increased the relative spleen weight as well as splenocyte proliferation induced by concanavalin A or bacterial lipopolysaccharide in mice. Natural killer (NK) cell activity in the HMWF-treated group was significantly increased compared to that in the control group. These results suggest that the HMWF of CW can support the immune system through secretion of macrophage cytokines, thereby enhancing NK cell activity and murine splenocyte proliferation.

• Journal of Veterinary Clinics
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• v.26 no.2
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• pp.138-143
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• 2009

Ischemia/reperfusion injury(I/RI) is the major cause of acute renal failure and delayed graft function(DGF) unavoidable in renal transplantation. Enormous studies on ischemia damage playing a role in activating graft rejection factors, such as T cells or macrophages, are being reported. Present study was performed to determine whether ischemia time would play an important role in activating rejection-related factors or not in rat models of I/RI. Male Sprague-Dawley rats were submitted to 30, 45, and 60 minutes of warm renal ischemia with nephrectomy or control animals underwent sham operation(unilateral nephrectomy). Renal function and survival rates were evaluated on day 0, 1, 2, 3, 5 and 7. Immunofluorescence staining of dendritic cells(DCs), natural killer(NK) cells, macrophages, B cells, CD4+ and CD8+ T cells were measured on day 1 and 7 after renal I/RI. Survival rates dropped below 50% after day 3 in 45 minutes ischemia. Histologic analysis of ischemic kidneys revealed a significant loss of tubular architecture and infiltration of inflammatory cells. DCs, NK cells, macrophages, CD4+ and CD8+ T cells were infiltrated from a day after I/RI depending on ischemia time. Antigen presenting cells(DCs, NK cells or macrophages) and even T cells were infiltrated 24 hours post-I/RI, which is at the time of acute tubular necrosis. During the regeneration phase, not only these cells increased but B cells also appeared in more than 45 minutes ischemia. The numbers of the innate and the adaptive immune cells increased depending on ischemia as well as reperfusion time. These changes of infiltrating cells resulting from each I/RI model show that ischemic time plays a role in activating rejection related immune factors and have consequences on progression of renal disease in transplanted and native kidneys.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.8
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• pp.919-928
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• 2017

The immuno-enhancing effects of alginate oligosaccharides from Sargassum coreanum were investigated. The alginate oligosaccharides were produced by an alginate-degrading enzyme from S. oneidensis PKA 1008. The degraded alginate oligosaccharides were visualized by thin-layer chromatography developed using a solvent system of 1-butanol/methanol/water, 4:1:2 (v/v/v). Alginate was degraded into dimmers at 60 h. As a result, the levels of Th1 cytokine [interferon $(IFN)-{\gamma}$ and interleukin (IL)-2] and Th2 cytokine (IL-6 and IL-10) increased with increasing incubation time compared to the control in vitro. Enzymatic extract treatment promoted proliferation of splenocytes at concentrations of 100 and 200 mg/kg at 24 h in vivo. Secretion of $IFN-{\gamma}$ and IL-2 significantly increased in a dose-dependent manner at 24 h as well as induced higher production of IgG2a in serum. Natural killer cell activity was measured and tended to increase. In addition, complete blood cell counts increased in a dose-dependent manner. These results indicate that alginate oligosaccharides produced by crude enzyme from S. oneidensis PKA 1008 may have significant immune activities.

• Clinical and Experimental Pediatrics
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• v.52 no.12
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• pp.1348-1357
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• 2009

Purpose:The aims of this study were to identify the clinical characteristics and determine the changes in the expression of cytokines and apoptosis-related genes in children with infectious mononucleosis. Methods:Serological examinations of 15 pediatric patients diagnosed with infectious mononucleosis were performed prospectively. Peripheral blood from the patients was used to compare the composition of T cell subsets, cytokines, Epstein-Barr virus (EBV) DNA, and the expression of apoptosis-related genes with those in 10 healthy children. Results:Mean age of the patient group was $5.7{\pm}3.4$ (range, 3-9) years, and the male-to-female ratio was 1.5:1. Fever, sore throat, pharyngitis/tonsillitis, and cervical lymph node enlargement were the most common symptoms and signs. The proportions of CD3+ T cells, CD8+ suppressor cells, and CD56+ natural killer (NK) cells were higher in the patient group than in the control group (P<0.01). The IL-2, IL-6, and interferon $(INF)-{\gamma}$ levels were higher in the early symptomatic period (P<0.01). Mean amount of EBV DNA in the patients was $10^{2.38}copies/{\mu}g$, and the amount was the highest at the beginning of the symptomatic period and normalized during the convalescent phase. Bcl-2 expression increased during the initial phase, while Bax expression increased during the convalescent phase. Further, FasL expression increased 1 week after symptom presentation and decreased during the convalescent phase. There was no significant change in Fas expression. Conclusion:We analyzed the clinical characteristics and changes in the expression ofcytokines and apoptosis-related genes in the patients with infectious mononucleosis.