• Title/Summary/Keyword: ELISA system

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Interleukin-5 Inhibition Assay of the Oriental Materia Medica Treatment by High Through Screening on the Splenocyte of Mouse (High through screening(HTS)를 이용한 백서(白鼠)의 비장세포(脾臟細胞)에서의 IL-5 분비 조절 한약물(韓藥物) Screening)

  • Park, Dong-Hee;Rhee, Hyung-Koo;Jung, Sung-Ki;Jung, Hee-Jae
    • The Journal of Internal Korean Medicine
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    • v.27 no.2
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    • pp.394-406
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    • 2006
  • Backgrounds and Objectives: Asthma is considered to be an inflammatory disease characterized by airway hyperresponsiveness and Pulmonary eosinophilia. And it is known the structure and function of IL-5, its receptor and the mechnism IL-5 triggered eosinophil accumulation and inflammaion of the airways. At this point of view, we assume which oriental materia medics can the splenocyte inhibit from secreting the IL-S in vitro. Material and Methosds: We used the splenocyte of mouse 8 weeks after its birth, and then cnltivated those into the 2 experimental groups and control group for 48 hours. The culture medium of experimental groups were made of $1{\mu}g/ml,\;10{\mu}g/ml$, oriental materia medics, representative. And the culture media of control group was given no oriental materia medica. Then, we assayed the quantity of cytokine-expression by the Sandwich ELISA. The quantifies of cytokine-expression of the experimental groups were compared with that of the control group which was standardized These method were used for the all of oriental materia medica treated. Results: In this study, we demonstrated that 12 oriental materia medica that inhibit the splenocyte from secreting the IL-5 in both $1{\mu}g/ml,\;and\;10{\mu}g/ml$ culture media. Those were Equiseti Herbs, Sophorae Subprostratae Radix, Moutan Radicis Cortex. Trichosanthis Radix, Buddleiae Flos. Cyperi Rhizoma. Benincasae Semen, Armeniacae Semen. Zedoariae Rhizoma, Astragali Semen, Dolichoris Semen. Lilii Bulbus, Asparagi Radix, Atractylodes Rhizoma White, Polygonati Officinallis Rhizoma. Conculusions: These findinga indicate that some oriental materia medica, specially Antipyretics, Herbs for Resolving Phlegm, Relieving Cough and Calming Wheezing and Herbs for Tonifring and Invigorating effects inhibit the splenocyte from secreting the IL-5. And further study experimented in vivo is needed for treating IL-5-driven inflammatory disease including asthma.

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Analysis of Monocyte Chemoattractant Protein 1(MCP-1) Polymorphism in Korean Patients with Asthma (한국인 천식환자의 Monocyte chemoattractant protein 1(MCP-1) 유전자 다형성에 대한 분석)

  • Hwang, Woo-Suk;Jeong, Seung-Yeon;Kim, Jin-Ju;Jung, Hee-Jae;Jung, Sung-Ki
    • The Journal of Internal Korean Medicine
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    • v.29 no.1
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    • pp.32-41
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    • 2008
  • Background : Monocyte chemoattractant protein-1(MCP-1), one of the CC chemokines, appears to play a significant role in asthma pathogenesis. It was reported that polymorphism in the MCP-1(-2518 A/G promoter) was associated with asthma in Caucasians, but the association of this polymorphism and asthma patients in the Korean population has not yet been clarified. Objective : We investigated the possible association between 2 polymorphisms (-2518 A/G promoter and Cys35Cys) and asthma patients in a Korean population. Materials and Methods : DNA samples were obtained from 86 Korean asthma patients and 270 healthy controls. MCP-1 genomic variants (-2518 A/G promoter and Cys35Cys polymorphism) were detected by PCR-RFLP. Level of MCP-1 was measured by ELISA for each genotype (n=8) (AA, AG, GG) and allele types of -2518 A/G promoter polymorphism for control subjects. Results : The Cys35Cys polymorphism was associated with asthma patients in Korean population [genotype distribution ($X^{2}=16.011$, P<0.001)]. Comparison of the two groups revealed no detectable differences in genotype and allele frequencies of the -2518 A/G polymorphism. Haplotype frequencies analysis revealed significant difference $(X^{2}=51.70$, P<0.001). MCP-1 serum level of subjects with G genotype of -2518 A/G promoter polymorphism was statistically higher than that with AA genotype (P<0.05). Conclusion : Our data indicate that no association exists between the MCP-1 -2518 A/G polymorphism and asthma susceptibility in the Korean population. However, it is noteworthy that the high prevalence of the -2518 G allele in the Korean population suggests a potentially important ethnic variation in the regulation of MCP-1 production. This variation must be considered in gene-association studies in different ethnic populations.

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Changes of Plasma and Urinary $TNF-{\alpha}$ in Children with Minimal Change Nephrotic Syndrome and Its Role in Albumin Permeability (미세변화신증후군 환아에서 Tumor Necrosis Factor-${\alpha}$의 혈중 및 요중 변화와 알부민 투과성에 미치는 영향)

  • Cho Min-Hyun;Lee Hwan-Seok;Oh Hyun-Hee;Chung Ki-Young;Koo Ja-Hoon;Ko Cheol-Woo
    • Childhood Kidney Diseases
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    • v.7 no.1
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    • pp.16-22
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    • 2003
  • Purpose : Minimal Change Disease(MCD) is the most common primary nephrotic syndrome in children. Some suggested that tumor necrosis factor-${\alpha}$ ($TNF-{\alpha}$) are involved in the pathogenesis of MCD. This study was done to see the changes of plasma and urinary $TNF-{\alpha}$, and their effects on the permeability of glomerular basement membrane. Methods : Study patients consisted of 19 biopsy-proven MCD children aged 2-15 years old. Both plasma and urinary $TNF-{\alpha}$ were measured. Employing the Millicell system, $TNF-{\alpha}$ were screened for the permeability factors. Results : Urinary $TNF-{\alpha}$ during relapse was significantly increased(P<0.01). No significant change was seen in the plasma $TNF-{\alpha}$ during relapse when compared to those in remission and the healthy controls. Furthermore, in the in vitro Millicell system, $TNF-{\alpha}$ did not produce a significant change in albumin permeability. Conclusion : Therefore, it seems that $TNF-{\alpha}$ may not play a disease-specific role in the pathogenesis of MCD.

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Anti-inflammatory Effect of Injinho-tang in RAW 264.7 Cells (마우스 대식세포인 RAW 264.7 세포에서 인진호탕(茵陳蒿湯)의 항염증 효과)

  • Yun, Hyun-Jeong;Heo, Sook-Kyoung;Yi, Hyo-Seung;Kim, Chang-Hyun;Kim, Byung-Wan;Park, Sun-Dong
    • The Korea Journal of Herbology
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    • v.23 no.2
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    • pp.169-178
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    • 2008
  • Objectives : Inflammation is important event in the development of vascular diseases including hypertension, atherosclerosis, and restenosis. Injinho-tang(IJHT) has been used as a traditional Korean herbal medicine since ancient times, and today it is widely used as a medication for jaundice associated with inflammation of the liver. The aim of this study was to determine whether IJHT and its components inhibit production of nitrite, an index of NO, and proinflammatory cytokines in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Methods : Cytotoxic activity of IJHT and its components on RAW 264.7 cells was using 5-(3-caroboxymeth-oxyphenyl)-2H-tetra-zolium inner salt (MTS) assay. The nitric oxide (NO) production was measured by Griess reagent system. And proinflammatory cytokines were measured by ELISA kit. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were detected by western blot. Results : IJHT and its components significantly inhibited the LPS-induced NO production and iNOS expression accompanied by an attenuation of tumor necrosis factor-alpha (TNF-${\alpha}$), interleukin-6 (1L-6), IL-$1{\beta}$ and monocyte chemoattractant protein-1 (MCP-1) formation in macrophages. Conclusions : IJHT and its components inhibit LPS-induced inflammation via decreasing cytokines production. These results indicate that IJHT and its components have potential as an anti-inflammation and anti-artherosclerosis agent.

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Effects of Cistanche Deserticola on Thyroid Function in Hypothyroidism Rat Model Induced by PTU(6-Propyl, 2-thiouracil) (육종용이 PTU로 유발된 갑상선 기능저하증 동물모델에 미치는 영향)

  • Lee, Seung-Jin;Paik, Sun-Ho;Ahn, Se-Young;Lee, Byung-Choel;Ahn, Young-Min
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.25 no.6
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    • pp.989-995
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    • 2011
  • Hypothyroidism is the most common disease in the endocrine system. Cistanche Deserticola has been used traditionally in the treatment of kidney deficiency and Yang deficiency syndrome. In this study, we investigated the therapeutic effects of Cistanche Deserticola on Hypothyroidism rat model induced by PTU. 24 Two-month-old Sprague-Dawley male rats were divided into 4 groups : 1) normal(n=6), 2) PTU-induced hypothyroidism control(n=6), 3) hypothyroidism rat treated with Cistanche Deserticola(n=6), 4) hypothyroidism rat treated with levothyroxine(n=6). PTU was administered for 4 weeks. Cistanche Deserticola and levothyroxine was administered 2 weeks after PTU was initiated for a total duration of 2 week. Body weights were checked every week and after 4 weeks, biochemical analysis was performed and T3, T4 and TSH were measured by ELISA kits. In comparison with the normal group, the control group showed hypothyroidism with lower T3, T4 level and higher TSH level. In the Cistanche group the level of T4 was significantly increased and TSH level was significantly decreased. There was no significant difference in biochemical labs and weight between the Cistanche group and the control group. These findings suggest that Cistanche Deserticola could help the production of thyroid hormones under PTU suppression. And there is no harmful effect on liver and kidney function, and other metabolism. According to these results Cistanche Deserticola could be an useful agent for treating hypothyroidism.

Highly Efficient Gene Expression in Rabbit Synoviocytes Using EBV-Based Plasmid (가토 윤활막 세포에서 EBV-Based 플라스미드를 사용한 효율적인 유전자 발현)

  • Kim, Jin Young;Oh, Sang Taek;Youn, JeeHee;Lee, Suk Kyeong
    • IMMUNE NETWORK
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    • v.4 no.3
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    • pp.190-197
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    • 2004
  • Background: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic synovial inflammation which leads to joint destruction. Gene therapy of RA targets the players of inflammation or articular destruction. However, viral vectors have safety problems and side effects, while non-viral vectors suffer from inefficient gene transfer and fast loss of gene expression. To overcome the limits of non-vial vectors, an EBV-based plasmid which is known to exert prolonged high level gene expression can be used. Methods: pEBVGFP, pEBVIL-10, and pEBVvIL-10 were constructed by cloning GFP, IL-10, and vIL-10 genes into an EBV-based plasmid, respectively. The pGFP was used as a control plasmid. Each constructs were lipofected into HIG-82 rabbit synoviocytes. The expression of GFP was monitored by FACS and confocal microscopy. IL-10 and vIL-10 expressions were measured by ELISA. Results: GFP expression 2 days after transfection was achieved in 33.2% of cells. GFP-expressing cells transfected with pGFP decreased rapidly from 4 days after transfection and disappeared completely by 11 days. Cells transfected with pEBVGFP began to decrease slowly from 4 days. But GFP expression was detected for over 35 days. In addition, HIG-82 cells transfected with pEBVIL-10 ($44.6{\pm}1.5ng/ml$) or pEBVvIL-10 ($51.0{\pm}5.7ng/ml$) secreted these cytokines at high levels. High level cytokine production by hygromycin selection was maintained at least for up to 26 days after transfection. Conclusion: These results suggest that the EBV-based plasmid has a potential to improve non-viral gene transfer system and may be applicable to treat RA without the drawbacks of viral vectors.

Serum Level of MMP-3 in Patients with Oral Squamous Cell Carcinoma - Lack of Association with Clinico-pathological Features

  • Tadbir, Azadeh Andisheh;Purshahidi, Sara;Ebrahimi, Hooman;Khademi, Bijan;Malekzadeh, Mahzad;Mardani, Maryam;Taghva, Masumeh;Sardari, Yasaman
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.9
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    • pp.4545-4548
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    • 2012
  • Background: MMP-3 is a proteolytic enzyme of the matrix metalloproteinase family. Protein degradation which is their fundamental action regulates different activities of tumor cell such as their growth, differentiation, apoptosis, migration, invasion, angiogenesis as well as their resistance to the immune system. Aim: The aim of this study was to determine MMP-3 serum levels in patients with OSCC and investigate if they correlate with clinicopathological features. Method and materials: Using an ELISA kit, we assessed and compared the circulating levels of MMP-3 in blood serum of 45 oral squamous cell carcinoma patients with 45 healthy control samples. Results: The serum MMP-3 level in OSCC patients was significantly higher ($9.45{\pm}4.6$ ng/ml) than healthy controls ($5.9{\pm}3.6$ ng/ml, p<0.001), especially in females and in older patients. However, there was no apparent correlation in serum MMP-3 concentration with the clinico-pathological features such as tumor location, stage, tumor size, nodal status, distant metastasis, histological grade and smoking. Discussion: This result suggests that the measurement of serum MMP-3 concentration might be helpful to diagnose OSCC but not to predict prognosis.

Anti-inflammatory effect of Salviae Miltiorrhizae Radix (단삼 (Salviae Miltiorrhizae Radix) 메탄올 추출물의 항염증 효과)

  • Yun, Hyun-Jeong;Heo, Sook-Kyoung;Yun, Hyung-Joong;Park, Won-Hwan;Park, Sun-Dong
    • The Korea Journal of Herbology
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    • v.22 no.4
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    • pp.65-73
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    • 2007
  • Objective : Salvia miltiorrhiza Bunge (Labiatae) (SM), an eminent herbal plant, has been widely used in traditional Chinese medicine for the treatment of vascular diseases such as hypertension. The aim of this study was to determine whether SM inhibits production of nitrite, an index of NO, and proinflammatory cytokines in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. And this study investigated whether or not SM could reduce tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$)-induced inflammatory response in human vascular aortic smooth muscle cells (HASMC) and umbilical vein endothelial cells (HUVEC). Methods : Cytotoxic activity of SM on RAW 264.7 cells was using 5-(3-caroboxymeth-oxy phenyJ)-2H-tetra-zolium inner salt (MTS) assay. We measured the NO production using Griess Reagent System. Production of Proliflammatory cytokines was measured by Enzyme-Linked Immunosorbent Assay (ELISA). Results : Our results indicated that SM significantly inhibited the LPS-induced NO production accompanied by an attenuation of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), IL-6 and monocyte chemoattractant protein (MCP)-1 formation in macrophages. SM decreased TNF-${\alpha}$-induced IL-8, IL-6 production, and intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression. Conclusion : These results indicate that SM has potential as an anti-inflammatory agent.

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Opposite Roles of B7.1 and CD28 Costimulatory Molecules for Protective Immunity against HSV-2 Challenge in a gD DNA Vaccine Model

  • Weiner, David B.;Sin, Jeong-Im
    • IMMUNE NETWORK
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    • v.5 no.2
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    • pp.68-77
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    • 2005
  • Background: Costimulation is a critical process in Ag-specific immune responses. Both B7.1 and CD28 molecules have been reported to stimulate T cell responses during antigen presentation. Therefore, we tested whether Ag-specific immune responses as well as protective immunity are influenced by coinjecting with B7.1 and CD28 cDNAs in a mouse HSV-2 challenge model system. Methods: ELISA was used to detect levels of antibodies, cytokines and chemokines while thymidine incorporation assay was used to evaluate T cell proliferation levels. Results: Ag-specific antibody responses were enhanced by CD28 coinjection but not by B7.1 coinjection. Furthermore, CD28 coinjection increased IgG1 production to a significant level, as compared to pgD+pcDNA3, suggesting that CD28 drives Th2 type responses. In contrast, B7.1 coinjection showed the opposite, suggesting a Th1 bias. B7.1 coinjection also enhanced Ag-specific Th cell proliferative responses as well as production of Th1 type cytokines and chemokines significantly higher than pgD+pcDNA3. However, CD28 coinjection decreased Ag-specific Th cell proliferative responses as well as production of Th1 types of cytokines and chemokine significantly lower than pgD+pcDNA3. Only MCP-1 production was enhanced by CD28. B7.1 coimmunized animals exhibited an enhanced survival rate as well as decreased herpetic lesion formation, as compared to pgD+pcDNA3. In contrast, CD28 vaccinated animals exhibited decreased survival from lethal challenge. Conclusion: This study shows that B7.1 enhances protective Th1 type cellular immunity against HSV-2 challenge while CD28 drives a more detrimental Th2 type immunity against HSV-2 challenge, supporting an opposite role of B7.1 and CD28 in Ag-specific immune responses to a Th1 vs Th2 type.

Immunization with a Genetically Engineered Uropathogenic Escherichia coli Adhesin-Escherichia coli Enterotoxin Subunit A2B Chimeric Protein

  • Lee, Yong-Hwa;Kim, Byung-O;Pyo, Suhk-Neung
    • Biomolecules & Therapeutics
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    • v.13 no.2
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    • pp.101-106
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    • 2005
  • The generation of secretory IgA antibodies (Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the heat-labile Escherichia coli enterotoxin A2B (ltxa2b) gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ltxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and westem blotting using antibodies to the maltose binding protein (MBP) or the heat labile E. coli subunit B (LTXB), plus the N-terminal amino acid sequence was analyzedd. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/LTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of LTXB. thisstudy also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA (sIgA) and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked LTXA2B acts as a useful mucosal adjuvant, and that adhesin/LTXA2A chimeric protein might be a potential antigen for oral immunization against UPEC.