Kim, Kyoung-Woon;Cho, Mi-La;Lee, Sang-Heon;Min, So-Youn;Park, Mi Kyung;Park, Sung-Hwan;Jue, Dae-Myung;Kim, Ho-Youn
IMMUNE NETWORK
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v.3
no.4
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pp.310-319
/
2003
Inflammatory mediators has been recognized as an important role in the pathogenesis of rheumatoid arthritis (RA). IL-17 is increasingly recognized as an important regulator of immune and inflammatory responses, including induction of proinflammatory cytokines and osteoclastic bone resorption. Evidence of the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA. However, the signaling pathways that regulate IL-17 production remain unknown. In the present study, we investigated the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in the regulation of IL-17 production in RA. PBMC were separated from RA (n=24) patients, and stimulated with various agents (anti CD3, anti CD28, PHA, ConA, IL-15). IL-17 levels were determined by sandwich ELISA and RT-PCR. The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody, PHA, IL-15 or MCP-1 (P<0.05). ConA also strongly induced IL-17 production (P<0.001), whereas TNF-alpha, IL-1beta, IL-18 or TGF-beta did not. IL-17 was detected in the PBMC of patients with osteoarthritis (OA) but their expression levels were much lower than those of RA PBMC. Anti-CD3 antibody activated the PI3K-Akt pathway and activation of the PI3K-Akt pathway resulted in a pronounced augmentation of nuclear factor kappaB ($NF-{\kappa}B$). IL-17 production by activated PBMC in RA is completely or partially blocked in the presence of $NF-{\kappa}B$ inhibitor PDTC and PI3K-Akt inhibitor, wortmannin and LY294002, respectively. Whereas the inhibition of AP-1 and extracellular signal-regulated kinase (ERK)1/2 did not affect IL-17 production. These results provide new insight into that PI3K/Akt and $NF-{\kappa}B$ dependent signal transduction pathway could be involved in the overproduction of key inflammatory cytokine, IL-17 in rheumatoid arthritis.
Kim, Yun-Sun;Kim, Eun-Young;Moon, Ji-Sook;Yoon, Tae-Ki;Lee, Woo-Sik;Lee, Kyung-Ah
Clinical and Experimental Reproductive Medicine
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v.38
no.4
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pp.193-202
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2011
Objective: We found previously that $interferon$$regulatory$ factor ($Irf$)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of $Irf-1$ in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of $Irf-1$ and the mouse oocyte maturation. Methods: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured $in$$vitro$ for 16 hours in the presence of varying concentrations of RA (0-10 ${\mu}M$). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 ${\mu}M$). With 100 nM RA treatment, lowest level of $Irf-1$ mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-${\alpha}$, macrophage inflammatory protein-$1{\beta}$, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. Conclusion: We concluded that the maturation of oocytes and $Irf-1$ expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes $in$$vitro$ by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for $in$$vitro$ oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.
Inflammation in the brain has known to be associated with the development of a various neurologiacal diseases. The hallmark of neuro-inflammation is the activation of microglia, brain macrophage. Pro-inflammatory compounds including nitric oxide(NO) are the main cause of neuro-degenerative disease such as Alzheimer's disease. In the study, we examined whether Harmonia axyridis extracts inhibit the NO production by a direct method using Griess reagent, western blotting and by RT-PCR(Reverse Transcription-Polymerase Chain Reactionin) the gene expression of inducible nitric oxide synthase(iNOS). Distilled water$(H_2O)$ and methanol(MeOH) extracts of H. axyridis inhibited the protein expression of TNF-a(Tumor Necrosis Factor) and IL-6(Interleukin) in LPS (Lipopolysaccharide) stimulated BV-2 cells at the concentration of 100 ng/ml. Incubation of BV-2 cells with the extracts of $H_2O$ of MeOH inhibited the LPS induced NO and iNOS protein. And this inhibition of iNOS protein is concordant with the inhibition of iNOS mRNA expression. These data suggested that H. axyridis extracts may play a crucial role in inhibiting the NO production.
Fragile histidine triad (FHIT) is a suppressor gene related to cervical cancer through CpG island hypermethylation. Folate is a water-soluble B-vitamin and an important cofactor in one-carbon metabolism. It may play an essential role in cervical lesions through effects on DNA methylation. The purpose of this study was to observe effects of folate and FHIT methylation and HPV 16 on cervical cancer progression. In this study, DNA methylation of FHIT, serum folate level and HPV16 status were measured using methylation-specific polymerase chain reaction (MSP), radioimmunoassay (RIA) and polymerase chain reaction (PCR), respectively, in 310 women with a diagnosis of normal cervix (NC, n=109), cervical intraepithelial neoplasia (CIN, n=101) and squamous cell carcinoma of the cervix (SCC, n=101). There were significant differences in HPV16 status (${\chi}^2=36.64$, P<0.001), CpG island methylation of FHIT (${\chi}^2=71.31$, P<0.001) and serum folate level (F=4.57, P=0.011) across the cervical histologic groups. Interaction analysis showed that the ORs only with FHIT methylation (OR=11.47) or only with HPV 16 positive (OR=4.63) or with serum folate level lower than 3.19ng/ml (OR=1.68) in SCC group were all higher than the control status of HPV 16 negative and FHIT unmethylation and serum folate level more than 3.19ng/ml (OR=1). The ORs only with HPV 16 positive (OR=2.58) or with serum folate level lower than 3.19ng/ml (OR=1.28) in CIN group were all higher than the control status, but the OR only with FHIT methylation (OR=0.53) in CIN group was lower than the control status. HPV 16 positivity was associated with a 7.60-fold increased risk of SCC with folate deficiency and with a 1.84-fold increased risk of CIN. The patients with FHIT methylation and folate deficiency or with FHIT methylation and HPV 16 positive were SCC or CIN, and the patients with HPV 16 positive and FHIT methylation and folate deficiency were all SCC. In conclusion, HPV 16 infection, FHIT methylation and folate deficiency might promote cervical cancer progression. This suggests that FHIT may be an effective target for prevention and treatment of cervical cancer.
Lee, Chang Youl;Ahn, Chul Min;Jeon, Jeong Hee;Kim, Hyung Jung;Kim, Se Kyu;Chang, Joon;Kim, Sung Kyu;Chang, Yoon Soo
Tuberculosis and Respiratory Diseases
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v.67
no.1
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pp.8-13
/
2009
Background: The insulin receptor substrate-1 (IRS-1) is the primary docking molecule for the insulin-like growth factor I receptor (IGF-IR), and is required for activation of the phosphatidylinositol 3'-kinase (PI3K) pathway. IRS-1 activation of the (PI3K) pathway regulates IGF-mediated survival, enhancement of cellular motility and apoptosis. Therefore, we attempted to ascertain whether IRS-1 genetic variations affect an individual's risk for non-small cell lung cancer (NSCLC). Methods: Two-hundred and eighteen subjects, either diagnosed with NSCLC or control subjects, were matched by age, gender and smoking status. Genomic DNA from each subject was amplified by PCR and analyzed according to the restriction fragment length polymorphism (RFLP) profile to detect the IRS-1 G972R polymorphism. Results: The frequencies of each polymorphic variation, in the control population, were as follows: GG=103 (94.5%) and GR=6 (5.5%); for the NSCLC subjects, the genotypic frequencies were as follows: GG=106 (97.2%) and GR=3 (2.8%). We could not demonstrate statistically significant differences in the genotypic distribution between the NSCLC and the control subjects (p=0.499, Fisher's Exact test). The relative risk of NSCLC, associated with the IRS-1 G972R polymorphic variation, was 1.028 (95% CI; 0.63~9.90). In addition, we found no differences between polymorphic variants with regard to the histological subtype of NSCLC. Conclusion: We did not observe any noteworthy differences in the frequency of the IRS-1 G972R polymorphism in NSCLC patients, compared to control subjects. These results suggest suggesting that, in our study population, the IRS-1 G972R polymorphism does may not appear to be associated with an increased risk of NSCLC.
Background: Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by diverse clinical manifestations and autoantibody production, which is known to be strongly influenced by genetic factors. Previous studies have revealed the associations of SLE with HLA class II alleles and antiphospholipid antibody system (anticardiolipin antibody (aCL) and anti-${\beta}_2$ glycoprotein I antibody (anti-${\beta}_2$ GPI)). Therefore, we studied the associations of HLA class II alleles with SLE and antiphospholipid antibody system. Methods: The genotyping for HLA-DRB1 and DQB1 alleles were performed in 61 SLE patients and 100 controls by the polymerase chain reaction (PCR)-sequence specific oligonucleotide probe method. ELISA tests for aCL and anti-${\beta}_2$ GPI were performed in 39 of the 61 SLE patients. The results were evaluated statistically by Chi-square test. Results: The frequencies of the HLA-$DRB1^*15$ and $DQB1^*06$ in SLE patients were significantly higher than those in controls. HLA-$DRB1^*12$ was significantly lower in SLE patients than controls. Nine of 39 patients were positive for aCL (IgG) and three were positive for aCL (IgM). One of 39 patients were positive for anti-${\beta}_2$ GPI (IgG) and none of them positive for anti-${\beta}_2$ GPI (IgM). Association of aCL with HLA class II alleles was not observed in our study. Conclusion: According to our results, it was found that HLA-$DRB1^*15$ and $DQB1^*06$ were associated with genetic susceptiblility and $DRB1^*12$ was associated with resistance to SLE in Korean population. No Association of aCL with HLA class II alleles was observed and the positive rate for anti-${\beta}_2$ GPI was very low.
Objectives : The purpose of this study is to investigate the effect of Ulmus davidiana Planch herbal acupuncture solution in LPS-stimulated RAW 264.7 cells and mouse knee joints, perfom1ed several experimental items: those are MIF mRNA, MIF, $TNF-{\alpha}$, $NF-{\kappa}B$ p65, iNOS mRNA, iNOS, NO, synovial hyperplasia, angiogenesis and fibrosis. Methods : In order to observe mRAN expression of MIF and iNOS in LPS-stimulated RAW 264.7 cells, RT-PCR was used. NO production in LPS-stimulated RAW 264.7 cells was measured by nitrite assay. All the female BALB/c mice were bred and maintained in pathogen-free mouse colonies and were 6 weeks of age on beginning of the experiment. The experimental model of RA was induced by injection of $50{\mu}g/kg$ LPS. Ulmus davidiana Planch herbal acupuncture solution was injected into either S 35 (犢鼻) or EX-LE 202 (內膝眼) of mice in turn daily for 19 days. Immunohistochemical staining was carried out to assess $TNF-{\alpha}$, $NF-{\kappa}B$ p65 and iNOS expression in synovial membrane. Synovial hyperplasia, angiogenesis and fibrosis in synovial membrane was observed with a microscope. Results : 1. Ulmus davidiana Planch herbal acupuncture solution inhibited mRNA expression of MIF and iNOS in dependence on a density of it in LPS-stimulated RAW 264.7 cells. 2. Ulmus davidiana Planch herbal acupuncture solution decreased synovial hyperplasia, angiogenesis and fibrosis in LPS-stimulated mouse knee joints. 3. Ulmus davidiana Planch herbal acupuncture solution curtailed production of MIF, $TNF-{\alpha}$, $NF-{\kappa}B$ p65, iNOS in LPS-stimulated mouse knee joints. Conclusion : On the basis of these results, It was shown that Ulmus davidiana Planch herbal acupuncture solution is significantly able to inhibit the production of MIF as a top in cytokines related to inflammatory or irrlll1une responses. Our results may provide that Ulmus davidiana Planch herbal acupuncture solution has beneficial effect in not only RA but other inflammatory or immune deases.
Kim, Su-Young;Chun, Hyung-Jon;Lee, Doo-Ik;Lee, Yun-Ho;Choi, Do-Young;You, Yong-Gu;Lee, Jae-Dong
Journal of Acupuncture Research
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v.24
no.1
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pp.137-159
/
2007
Objectives: The purpose of this study is to examine the risk factors and the genetic polymorphism of TNF-alpha associated with rheumatoid arthritis by Sasang constitution Methods : This study was planned to detect the susceptibility of the patients diagnosed by rheumatoid arthritis to Sasang Constitution and to examine the risk factor such as life style and environmental stress (smoking, environmental tobacco smoke, alcohol intake and so on). The genetic polymorphism of TNF-alpha (G308A) were analyzed by PCR-RFLP in rheumatoid arthritis patients and controls. Rheumatoid arthritis patients and matched controls are assessed with QSCCII question for Sasang Typology. Then the genetic polymorphism of patients by Sasang constitution are compared to those of control, which are statistically analyzed and adjusted by age, sex, smoking status, alcohol intake, BMI, and econocmic status. Results: Differential effect of passive smoking on the association between Sasang constitution and rheumatoid arthritis risk was found. This study showed that the genetic polymorphism (TNF-${\alpha}$(G308A)) of rheumatoid arthritis patients and controls associated with the susceptibility to rheumatoid arthritis by sasang constitution was analyzed. Differential effects of TNF-${\alpha}$(G308) genetic polymorphism on the association between rheumatoid arthritis risk and Sasang constitution were found. Conclusion : It is suggested that the genetic polymorphism correlated with susceptibility to rheumatoid arthritis by specific sasang constitution used as its susceptibility marker and further as basic data to prevent the risk factors for rheumatoid arthritis. But larger studies will be needed to confirm these preliminary findings.
It is commonly acknowledged that bone morphogenic protein (BMP-2) functions as a potential osteogenic inducer in bone formation. Recently, several papers reported that bone marrow-derived stem cell (BMSC) from human is not responsive to BMP-2 in comparison to high capacity of BMP-2 in the osteoinduction of stromal cell derived from bone marrow of rodent animals such as rat or mouse. In this study, we characterized BMSC derived from 11 years old donor for the responsiveness to rhBMP-2, dexamethasone (Dex) and 1,25-dihydroxyvitamin D (vitamin D), in order to analyze their function in the early osteogenesis. The effect of over mentioned agents was evaluated by means of assessing alkaline phosphatase (ALP) activity/staining, RT-PCR analysis and von Kossa staining. In addition, we analyzed the meaning of expressed several osteoblastic markers such as alkaline phosphatase, collagen typeI, osteopontin, bone sialoprotein and osteocalcin with relation to either differentiation or mineralization. Only in the presence of Dex, human BMSC could commit osteoblastic differentiation and matrix mineralization, and either BMP-2 or vitamin D treatment was not able to induce. But BMP-2 or Vitamin D showed potential synergy effect with Dex. ALP and bone sialoprotein were clearly expressed in response of Dex treatment compared to weak expression of osteopontin in early osteogenesis. Therefore, we expect that this study will contribute partly to elucidiating early osteogenesis mechanism in human, but variations among bone marrow donors must be considered through further study.
Bone remodeling is a process controlled by the action of two major bone cells; the bone forming osteoblast and the bone resorbing osteoclast. In the process of osteoclastogenesis, stromal cells and osteoblast produce RANKL, OPG, and M-CSF, which in turn regulate the osteoclastogenesis. During the bone resorption by activated osteoclasts, extracellular $Ca^{2+}/{PO_4}^{2-}$ concentration and degraded organic materials goes up, providing the hypertonic microenvironment. In this study, we tested the effects of hypertonicity due to the degraded organic materials on osteoclastogenesis in co-culture system. It was examined the cellular response of osteoblastic cell in terms of osteoclastogenesis by applying the sucrose, and mannitol, as a substitute of degraded organic materials to co-culture system. Apart from the sucrose, mannitol, and NaCl was tested to be compared to the effect of organic osmotic particles. The addition of sucrose and mannitol (25, 50, 100, 150, or 200 mM) to co-culture medium inhibited the number of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells induced by 10 nM $1{\alpha},25(OH)_2vitaminD_3$ ($1{\alpha},25(OH)_2D_3$). However, NaCl did exert harmful effect upon the cells in this co-culture system, which is attributed to DNA damage in high concentration of NaCl. To further investigate the mechanism by which hypertonicity inhibits $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis, the mRNA expressions of receptor activator of nuclear factor (NF)-kB ligand (RANKL) and osteoprotegerin (OPG) were monitored by RT-PCR. In the presence of sucrose (50 mM), RANKL mRNA expression was decreased in a dose-dependent manner, while the change in OPG and M-CSF mRNA were not occurred in significantly. The RANKL mRNA expression was inhibited for 48 hours in the presence of sucrose (50 mM), but such a decrement recovered after 72 hours. However, there were no considerable changes in the expression of OPG and M-CSF mRNA. Conclusively, these findings strongly suggest that hypertonic stress down-regulates $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis via RANKL signal pathway in osteoblastic cell, and may playa pivotal role as a regulator that modulates osteoclastogenesis.
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