• Journal of Acupuncture Research
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• v.22 no.3
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• pp.155-167
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• 2005

Objectives : The purpose of this study was to investigate the anti-inflammatory effect of Cobrotoxin on binding affinity of cobrotoxin with P50, $IKK{\alpa}$ and $IKK{\beta}$, activities of NF-${\kappa}B$, Cell viability of astrocyte, expressions of protein molecules of NF-${\kappa}B$ such as P50, P-$1{kappa}B$, $1{\kappa}B$ and iflammation related genes such as Cox-2, iNOS, cPLA2 in the SNP or LPS induced Inflammatory pathway of Rats' astrocytes. Methods : In this study, The expression of cytosolic phospholipase A2, Nitric oxcide, Cyclooxygenase-2 and inducible nitrogen oxide synthase was determined by western blotting with corresponding antibodies, and the generation of NF-${\kappa}B$ was assayed by EMSA method in astrocytes of rats. The Cell viability of astrocytes was determined by MTT assay, and Binding affinity of Cobrotoxin with P50, $IKK{\alpha}$ and $IKK{\beta}$ was assayed by Surface plasmon resonance analysis, and NF-${\kappa}B$ dependent luciferase activity was determined by luciferase analysis, and Uptake of cobrotoxin in astrocytes was identified by Confocal laser scanning microscope Results : 1. Compared with control, LPS-induced NF-${\kappa}B$ DNA binding activity was decreased significantly by 0.1, $0.5{\mu}g/m{\ell}$ of Cobrotoxin in Astrocyte. 2. Compared with control, LPS-induced NF-kB dependent luciferase expression was decreased significantly by 0.1, 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin in Astrocyte. 3. Compared with control, SNP induced P50, $I{\kappa}B$ expressions in astrocyte were decreased significantly by 0.1, 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin and P-$1{\kappa}B$ expression was decreased significantly by 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin. 4. Compared with control, LPS induced P50, $1{\kappa}B$ expressions in astrocyte were decreased significantly by 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin. 5. Compared with control, SNP induced Cox-2, iNOS, CPLA2 expressions in astrocyte were decreased significantly by $1{\mu}g/m{\ell}$ of Cobrotoxin. 6. Compared with control, LPS induced Cox-2, cPLA2 expressions in astrocyte were decreased significantly by 0.1, 0.5, $1{\mu}g/m{\ell}$ of Cobrotoxin and iNOS expression was decreased significantly by 0.5, $1{\mu}g/m{\ell}$ of Cobrotoxin. 7. Compared with $0.5{\mu}g/m{\ell}$ of Cobrotoxin, SNP-induced NF-${\kappa}B$ DNA bindins activity in astrocyte was increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM. 8. Compared with $0.5{\mu}g/m{\ell}$ of Cobrotoxin, LPS-induced NF-${\kappa}B$ DNA binding activity in astrocyte was increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM, Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM, Cobrotoxin $0.5{\mu}g/m{\ell}$with GSH 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 5mM 9. Compared with $0.1{\mu}g/m{\ell}$ of cobrotoxin, SNP induced P50 expressions in astrocyte were increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM, Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 5mM. 10. The uptake of the labeled cobrotoxin into the cells was shown under a confocal laser scanning microscope. cobrotoxin was uptaken into the membrane and nucleus of astrocytes. Conclusions : In summary, the present results demonstrate that cobrotoxin directly binds to sulfhydryl group of p50 and IKKS resulting In the reduction of translocation of p50 and IkB release, thereby inhibits activation of NF-${\kappa}B$, and suggest that pico to nanomolar range of cobrotoxin could inhibit the expression of genes in the NF-${\kappa}B$ signal pathway.

• Tuberculosis and Respiratory Diseases
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• v.42 no.4
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• pp.569-583
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• 1995

Background: The expression of the adhesion molecules on the cell surface is important in the movement of cells and the modulation of immune response. DILD starts as an alveolitis and progresses to pulmonary fibrosis. So adhesion molecules in these patients is expected to be increased. There are several reports about adhesion molecules in DILD in terms of the percentage of positive cells in immuno-stain, in which the interpretation is subjective and the data were variable. Methods: So we measured the relative median fluorescence intensity(RMFI) which is the ratio of the FI emitted by bound primary monoclonal antibody to FI emitted by isotypic control antibody of the cells in BALF of 28 patients with DILD(IPF:10, collagen disease:7, sarcoidosis:9, hypersensitivity pneumonitis:2) and 9 healthy control. Results: RMFI of the ICAM-1 on AM($3.30{\pm}1.16$) and lymphocyte($5.39{\pm}.70$) of DILD were increased significantly than normal control($0.93{\pm}0.18$, $1.06{\pm}0.21$, respectively, p=0.001, P=0.003). RMFI of the CD18 on lymphocyte was also higher($24.9{\pm}14.9$) than normal($4.59{\pm}3.77$, p=0.0023). And there was a correlation between RMFI of ICAM on AM and the % of AM(r=-0.66, p=0.0001) and lymphocyte(r=0.447, p=0.0116) in BALF. Also RMFI of ICAM on lymphocyte had a significant (r=0.593, p=0.075) correlation with the % of IL-2R(+) lymphocyte in BALF. The soluble ICAM(sICAM) in serum was also significantly elevated in DILD($499.7{\pm}222.2\;ng/ml$) compred to normal($199.0{\pm}38.9$) (p=0.00097) and sICAM in BAL fluid was also significantly higher than normal control group($41.8{\pm}23.0\;ng/ml$ vs $20.1{\pm}13.6\;ng/ml$). There was a Significant correlation between sICAM level in serum and the expression of ICAM-l on AM(r=0.554, p=0.0259).Conclusion: These data suggest that in DILD the expression of adhesion molecules is increased in the AM and BAL lymphocytes with elevated serum sICAM, and these parameter may be useful in determining disease activity.

• Journal of Life Science
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• v.16 no.6
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• pp.904-910
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• 2006

Dendritic cells (DCs) are the only antigen presenting cells (APCs) capable of initiating immune responses, which is crucial for priming the specific cytotoxic T lymphocyte (CTL) response and tumor immunity. Upon activation by DCs, CD4+ helper T cells can cross-prime CD8+ CTLs via IL-12. However, recently activated DCs were described to prime in vitro strong T helper cell type 1 $(Th_1)$ responses, whereas at later time points, they preferentially prime $Th_2$ cells. Therfore, we examined in this study the optimum kinetic state of DCs activation impacted on in vivo priming of tumor-specific CTLs by using ovalbumin (OVA) tumor antigen model. Bone-marrow-derived DCs showed an appropriate expression of surface MHC and costimulatory molecules after 6 or 7-day differentiation. The 6-day differentiated DCs pulsed with OVA antigen for 8 h (8-h DC) and followed by restimulation with LPS for 24 h maintained high interleukin (IL)-12 production potential, accompanying the decreased level in their secretion by delayed re-exposure time to LPS. Furthermore, immunization with 8-h DC induced higher intracellular $interferon(IFN)-{\gamma}+/CD8+T$ cells and elicited more powerful cytotoxicity of splenocytes to EG7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with 24-h DC. In the animal study for the evaluation of therapeutic or protective antitumor immunity, immunization with 8-h DC induced an effective antitumor immunity against tumor of EG7 cells and completely protected mice from tumor formation and prolonged survival, respectively. The most commonly used and clinically applied DC-based vaccine is based on in vitro antigen loading for 24 h. However, our data indicated that antigen stimulation over 8 h decreased antitumor immunity with functional exhaustion of DCs, and that the 8-h DC would be an optimum activation state impacted on in vivo priming of tumor-specific CTLs and subsequently lead to induction of strong antitumor immunity.

• Journal of Life Science
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• v.28 no.10
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• pp.1220-1232
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• 2018

The purpose of this animal study was to evaluate, by histological analysis, bone regeneration in rabbit maxillary sinuses with an anorganic bovine graft (Bio-Oss) and a ${\beta}-tricalcium$ phosphate (${\beta}-TCP$) grafting. Bilateral sinus augmentation procedures were performed in 12 adult male rabbits. Rectangular replaceable bony windows were made with a piezoelectric thin saw insert. In the Bio-Oss group, Bio-Oss was grafted and in the ${\beta}-TCP$ group, ${\beta}-TCP$ was grafted and covered by replaceable bony windows. The animals were sacrificed at 2, 4, and 8 weeks after the surgical procedure. The augmented sinuses were evaluated by histomorphometric analysis using hematoxylin-eosin, Masson trichrome, and tartrate-resistant acid phosphatase stains and also by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), type I collagen, and osteocalcin content. Histologically, new bone formation was found on the surface of Bio-Oss and ${\beta}-TCP$ particles from 2 weeks and continued to 8 weeks. Significant higher new bone formation was revealed in the ${\beta}-TCP$ group than in the Bio-Oss group at 8 weeks. The amount of graft materials was significantly decreased in the ${\beta}-TCP$ group and the number of osteoclasts was significantly increased in the ${\beta}-TCP$ group from 4 to 8 weeks. Immunoreactivity to PCNA was reduced at 8 weeks. The expression of type I collagen was significantly increased in the ${\beta}-TCP$ group at 2 weeks, but was significantly increased in the Bio-Oss group at 8 weeks. Immunoreactivity to osteocalcin was increased from 2 to 8 weeks. These histological results can help in the selection of graft materials for implants. Both Bio-Oss and ${\beta}-TCP$ are proven graft materials, however, these results indicate that ${\beta}-TCP$ showed better bone regeneration results in rabbit maxillary sinus augmentation.

• Clinical and Experimental Pediatrics
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• v.46 no.3
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• pp.271-276
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• 2003

Purpose : Granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF) are principal cytokines in granulopoiesis and their physiologic effects are mediated through binding to specific cell surface receptors. Although it is known that the level of serum G-CSF and GM-CSF, and presentation of the receptors are increased in infectious diseases, there have been no studies to find the correlation between the granulopoiesis and leukocytosis. This study was designed to measure G-CSF and GM-CSF in leukocytosis and in control and to demonstrate the possible pathogenesis of granulopoiesis in leukocytosis using quantitative analysis of G-CSF, GM-CSF and their CSFr. Methods : The plasma levels of G-CSF, GM-CSF of 13 children without leukocytosis and 14 children with leukocytosis were measured. Counts of cell surface G-CSFr and GM-CSFr were measured by combining anti G-CSFr and anti GM-CSFr monoclonal antibodies to their respective receptors by using quantitative flow cytometric assay. Results : There was no significant difference betweeen the plasma concentration of G-CSF and GM-CSF in acute leukocytosis and in the control group. However, levels of G-CSFr in acute leukocytosis decreased significantly compared to the control(P=0.012) and the levels of GM-CSFr in both groups revealed no significant difference. Conclusion : Increase in the number of leukocyte in leukocytosis was mediated by increasing the number of neutrophil, and increased plasma concentration of G-CSF may be the cause of neutrophilia. But GM-CSF did not have any influence on leukocytosis.

• Korean Journal of Poultry Science
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• v.28 no.3
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• pp.239-244
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• 2001

To examine the effects of feed additives on the expression of Perpheral blood cell surface molecules, phagocytosis and antigen specific antibody formation, broilers were randomly assigned to T$_1$, T$_2$, T$_3$, and T$_4$ groups. T$_1$ group was fed diet without any additives for 13 weeks, T$_2$ was fed diet with full fat flax, T$_3$ was fed diet with full fat flax containing $\alpha$-tocopherol, and T$_4$ was fed diet with full-fat flax containing $\alpha$-tocopherol and selenium. Since 5 weeks feeding the data were examined by luminometer. After 2 weeks adminstration of different feeding, although all treated groups (T$_2$, T$_3$, and T$_4$,) showed slightly increased chemiluminescence (CL) responses than T$_1$, this result was not significant. After 4 weeks feeding there was no significant increase of CL in the Phagocytes like neutrophils and macrophages of T$_2$ group compared to T$_1$. But phagocytes from T$_3$ and T$_4$ group showed in creased $O_2$- (6%, 18% respectively) as well as $H_2O$$_2$ (9.5% and 10.9%, respectively) induced CL responses. After 8 weeks feeding there was more than 50% increase $O_2$- induced CL in T$_3$ and T$_4$ group, but $H_2O$$_2$ induced CL responses in T$_3$ and T$_4$ group was slightly increased (6.6% and 9.3%, respectively).

• Tuberculosis and Respiratory Diseases
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• v.52 no.5
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• pp.497-505
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• 2002

Background : INF-${\gamma}$ plays an important role in the host response to a mycobacterial infection. A complete IFN-${\gamma}$ receptor 1 deficiency is a life threatening condition because it renders patients highly susceptible to a mycobacterial infection. Several mutations in the IFN-${\gamma}$ receptor and STAT1 gene have been identified in the rare mycobacterial infections. These mutations have partial function of the IFN-${\gamma}$ receptor and similar pathologic features to clinical tuberculosis. Materials and Methods : The function of the IFN-${\gamma}$ receptor was evaluated in the patients with clinical tuberculosis. In addition, the DNA coding sequence of the IFNgR1 and STAT1 gene was also analyzed in disseminated tuberculosis patients who might have a defective IFN-${\gamma}$ receptor. Results : The cell surface expression levels of HLA-DR and CD64 in the PMBC after being stimulation with IFN-${\gamma}$ (100IU/ml, 1000IU/ml) were increased in both controls and patients. However, the rate of increase in both groups was similar. The production of TNF-${\alpha}$ in the response to stimulation with LPS was higher in the both groups ($850.7{\pm}687.8$ vs. $836.7{\pm}564.3$ pg/ml). Pretreatment with IFN-${\gamma}$ prior to LPS stimulation resulted in further increase in TNF-${\alpha}$ production between both groups ($2203.5{\pm}242.5$ vs. $2227.5{\pm}560.4$ pg/ml). However, the rate of the increase in TNF-${\alpha}$ production in the both groups was similar. The known mutations in the IFNgR1 and STAT1 coding sequences were not found in the genomic DNA of patients with disseminated tuberculosis. Conclusion : The functional and genetic defects of the IFN-${\gamma}$ receptor were not identified in clinical tuberculosis. This suggests the defective IFN-${\gamma}$ receptor that predispoe patients to a BCG or NTM infection can not alone account for the cases of clinical tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.45 no.4
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• pp.846-860
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• 1998

Background: Endotoxin (LPS : lipopolysaccharide), a potent activator of immune system, can induce acute and chronic inflammation through the production of cytokines by a variety of cells, such as monocytes, endothelial cells, lymphocytes, eosinophils, neutrophils and fibroblasts. LPS stimulate the mononucelar cells by two different pathway, the CD14 dependent and independent way, of which the former has been well documented, but not the latter. LPS binds to the LPS-binding protein (LBP), in serum, to make the LPS-LBP complex which interacts with CD14 molecules on the mononuclear cell surface in peripheral blood or is transported to the tissues. In case of high concentration of LPS, LPS can stimulate directly the macrophages without LBP. We investigated to detect the generation of proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF-$\alpha$ and fibrogenic cytokine, TGF-$\beta$, by peripheral blood mononuclear cells (PBMC) after LPS stimulation under serum-free conditions, which lacks LBPs. Methods : PBMC were obtained by centrifugation on Ficoll Hypaque solution of peripheral venous bloods from healthy normal subjects, then stimulated in the presence of LPS (0.1 ${\mu}g/mL$ to 100 ${\mu}g/mL$ ). The activities of IL-1, IL-6, TNF, and TGF-$\beta$ were measured by bioassaies using cytokines - dependent proliferating or inhibiting cell lines. The cellular sources producing the cytokines was investigated by immunohistochemical stains and in situ hybridization. Results : PBMC started to produce IL-6, TNF-$\alpha$ and TGF-$\beta$ in 1 hr, 4 hrs and 8hrs, respectively, after LPS stimulation. The production of IL-6, TNF-$\alpha$ and TGF-$\beta$ continuously increased 96 hrs after stimulation of LPS. The amount of production was 19.8 ng/ml of IL-6 by $10^5$ PBMC, 4.1 ng/mL of TNF by $10^6$ PBMC and 34.4 pg/mL of TGF-$\beta$ by $2{\times}10^6$ PBMC. The immunoreactivity to IL-6, TNF-$\alpha$ and TGF-$\beta$ were detected on monocytes in LPS-stimulated PBMC. Some of lymphocytes showed positive immunoreactivity to TGF-$\beta$. Double immunohistochemical stain showed that IL-1$\beta$, IL-6, TNF-$\alpha$ expression was not associated with CD14 postivity on monocytes. IL-1$\beta$, IL-6, TNF-$\alpha$ and TGF-$\beta$mRNA expression were same as observed in immunoreactivity for each cytokines. Conclusion: When monocytes are stimulated with LPS under serum-free conditions, IL-6 and TNF-$\alpha$ are secreted in early stage of inflammation. In contrast, the secretion of TGF-$\beta$ arise in the late stages and that is maintained after 96 hrs. The main cells releasing IL-1$\beta$, IL-6, TNF-$\alpha$ and TGF-$\beta$ are monocytes, but also lymphocytes can secret TGF-$\beta$.