• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2635-2640
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• 2014

Sera of cancer patients may contain antibodies that react with a unique group of autologous cellular antigens called tumor-associated antigens (TAAs). The present study aimed to determine whether a mini-array of multiple TAAs would enhance antibody detection and be a useful approach in esophageal cancer detection and diagnosis. Our mini-array of multiple TAAs consisted of eleven antigens, p53, pl6, Impl, CyclinB1, C-myc, RalA, p62, Survivin, Koc, CyclinD1 and CyclinE full-length recombinant proteins. Enzyme-linked immunosorbent assays (ELISA) were used to detect autoantibodies against eleven selected TAAs in 174 sera from patients with esophageal cancer, as well as 242 sera from normal individuals. In addition, positive results of ELISA were confirmed by Western blotting. In a parallel screening trial, with the successive addition of antigen to a final total of eleven TAAs, there was a stepwise increase in positive antibody reactions. The eleven TAAs were the best parallel combination, and the sensitivity and specificity in diagnosing esophageal cancer was 75.3% and 81.0%, respectively. The positive and negative predictive values were 74.0% and 82.0%, respectively, indicating that the parallel assay of eleven TAAs raised the diagnostic precision significantly. In addition, the levels of antibodies to seven antigens, comprising p53, Impl, C-myc, RalA, p62, Survivin, and CyclinD1, were significantly different in various stages of esophageal cancer, which showed that autoantibodies may be involved in the pathogenesis and progression of esophageal cancer. All in all, this study further supports our previous hypothesis that a combination of antibodies might acquire higher sensitivity for the diagnosis of certain types of cancer. A customized mini-array of multiple carefully-selected TAAs is able to enhance autoantibody detection in the immunodiagnosis of esophageal cancer and autoantibodies to TAAs might be reference indicators of clinical stage.

• Journal of the korean academy of Pediatric Dentistry
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• v.33 no.4
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• pp.587-596
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• 2006

Most of oral streptococci express the Antigen I/II (AgI/II) proteins, cell wall anchored adhesions. AgI/II protein binds to salivary agglutinin glycoprotein, a component of tooth pellicle and to ligands in other bacteria. These associations play important roles in bacterial colonization. Recently, it was reported that diverse host molecules also interact with AgI/II protein and that these interactions induce inflammatory responses from host cells. Among mutans streptococci containing -type hemolytic activity, Streptococcus mutans is a causative agent for dental caries. Compared with many other strains of S. mutans, GS-5 strain is unique in that this bacterium expresses truncated secretory AgI/II protein due to the nonsense mutation in the agI/II gene. This indicates that S. mutans GS-5 has a different clinical role and a recent report supported this idea based on the results from clinically isolated S. mutans strains. Previously, we had cloned agI/II gene from S. mutans GS-5 and generated recombinant N-terminal AgI/II protein. In this study, we further produced a hybridoma line expressing anti-AgI/II monoclonal antibodies named as 1C11A. This antibody showed high sensitivity to AgI/II protein in Western blot and ELISA. This new reagent will provide a basis for investigating the mechanisms of AgI/II-related diseases.

• The Journal of Korean Society of Virology
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• v.26 no.1
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• pp.77-90
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• 1996

Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the virus in the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western blotting. Therefore, the preparation of this protein in the level of higher quantity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA) virus and expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen. First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase $H^-$ AMV reverse transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplified specifically by Taq DNA polymrerase. The pT7blue (R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of BL21 (DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression. Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG was used and the expressed protein was characterized by Western blotting. The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from patients of HFRS as to be tested by ELISA and Western blotting.

• Parasites, Hosts and Diseases
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• v.37 no.3
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• pp.163-169
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• 1999

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophogoides pteronyssinus, were produce. Four monoclonal antibodies produced wee species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides, A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p2 in house dust. The sensitivity limit was 4ng/ml with rDer p2 and $8{\;}\mu\textrm{g}/ml$ with the d. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p2 could be useful for the environmental studies and for the standardization of mite allergen extracts.

• The Journal of Pediatrics of Korean Medicine
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• v.28 no.2
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• pp.23-39
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• 2014

Objectives PRAL (Prunus armniaca Linne Var) is a herbal formula in Oriental Medicine, known for its anti-inflammatory and anti-allergenic properties. However, its mechanism of action and the cellular targets have not yet been found enough. The purpose of this study is to investigate the effects of PRAL on Th2 cytokines expression in MC/9 mast cells. Methods The effect of PRAL was analyzed by ELISA, Real-time PCR, Western blot in MC/9 mast cells. mRNA levels of GM-CSF, IL-4, IL-5, IL-6, IL-13, TNF-${\alpha}$ were analyzed with Real-time PCR. Levels of IL-13, MIP-$1{\alpha}$ were measured using enzyme-linked immunosorbent assays (ELISA). NFAT, AP-1 and NF-${\kappa}B$ p65 were examined by Western blot analysis. Results PRAL inhibited GM-CSF, IL-4, IL-5, IL-6, IL-13, TNF-${\alpha}$ mRNA expression in a dose dependent manner. GM-CSF, IL-4, IL-5 mRNA expression were inhibited significantly in comparison to DNP-IgE control group at concentration of 100 ${\mu}g/ml$ and IL-6, IL-13, TNF-${\alpha}$ mRNA expression were inhibited at concentration of 50 ${\mu}g/ml$, 100 ${\mu}g/ml$. PRAL also inhibited the IL-13, MIP-$1{\alpha}$ production significantly in comparison to DNP-IgE control group in a dose dependent manner. IL-13 production was inhibited at a concentration of 200 ${\mu}g/ml$, 400 ${\mu}g/ml$ and MIP-$1{\alpha}$ was inhibited at a concentration of 100 ${\mu}g/ml$, 200 ${\mu}g/ml$, 400 ${\mu}g/ml$. Western blot analysis of transcription factors involving Th2 cytokines expression revealed prominent decrease of the mast cell specific transcription factors including NFAT-1, c-Jun as well as NF-${\kappa}B$ p65 but not NFAT-2 and c-Fos. Conclusion These results indicate that PRAL has the effect of suppressing Th2 cytokines production in the MC/9 mast cells. These data represent that PRAL potentiates therapeutic activities to the allergic disease by regulating Th2 cytokines in the MC/9 mast cells.

• Restorative Dentistry and Endodontics
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• v.26 no.6
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• pp.451-463
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• 2001

목적 - Inflammatory cytokine으로 알려진 interleukin 1$\beta$, tumor necrosis factor $\alpha$는 치수 및 치근단질환에서 주요한 역할을 하며, 골흡수를 자극하고 골형성을 방해하는 것으로 알려져 왔다. 이들 cytokine은 주로 단핵세포/대식세포가 형성하는 것으로 알려져 왔으나 최근 연구에 의하면, PMN도 또한 이 런 cytokine들을 형성할 수 있다는 것이 보고되었다. 오랫동안 염증반응이나 면역반응에서 PMN의 역할이 주로 포식작용 을 통해 병원균을 제거하는 것이라고만 생각되어져 왔던 것을 생각하면, 새로운 발견이라 할 수 있다. 또, PMN은 IL-1ra도 생성하는 것으로 보고되었는데, IL-1ra란 IL-1의 생물학적 작용을 방해하는 인자이므로, IL-1과 밀접한 관련을 가지는 질환의 발전에 있어서 IL-1과 IL-1ra의 balance가 매우 중요한 역할을 할 것으로 생각된다 즉, IL-1ra는 IL-1$\beta$의 proinflammatory effect를 제한할 수 있는 negative feedback mechanism이라고 할 수 있다. 이 연구의 목적은 치수 및 치근단 조직의 감염에 있어서 주요 원인균인 Porphyromonas endodontalis의 LPS가 PMN의 IL-1$\beta$, TNF-$\alpha$, IL-1ra생성에 미치는 영향을 단백질과 mRNA 수준에서 관찰하는 것이다. 잘 알려진 non-oral bacterium인 E. coli의 LPS를 positive control로 사용하였으며, IL-1ra가 IL-1$\beta$의 생물학적 작용을 방해하는 작용을 관찰하기 위해, IL-1의 biological assay도 시행하였다. 방법 - P. endodontalis ATCC 35406을 혐기성 조건에서 배양하고, hot phenol-water extraction의 방법으로 LPS를 추출(crude LPS)한 후, 제조회사로부터 구입한 E. coli의 crude LPS와 함께 정제하였다. 건강한 자원자들을 대상으로 말초혈액을 채취한 후 dextran sedimentaion을 거쳐 Lymphoprep을 이용하여 PMN층을 분리하였다. 얻어진 세포들은 RPMI 1640 (supplemented with fetal bovine serum antibiotics)에 5$\times$$10^{6}$cells/ml이 되도록 resuspend시킨 후 각기 다른 농도 (0, 0.01, 0.1, 1 and 10$\mu$g/ml)의 LPS를 처리하여, 각기 다른 시간(Northern blot : 1, 2, 4시간 ELISA : 2, 6, 12, 18시간)동안 37$^{\circ}C$ in 5% $CO_2$ 의 조건으로 배양하였다. 상층액은 -7$0^{\circ}C$에 보관하였다가 추후에 ELISA를 이용한 단백질 농도 측정과 IL-1 biological assay에 사용되어졌으며, 배양된 세포로부터 RNA를 추출하여 Northern hybridization을 통해 mRNA expression을 관찰하였다. (중략)

• Journal of fish pathology
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• v.26 no.3
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• pp.241-254
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• 2013

In order to establish bio-marker systems for the screening of endocrine-disrupting chemicals contaminated in various environment, Vitellogenin(Vtg) bio-marker have been developed to detect Scorpion fish's(Sebastiscus marmoratus) Vtg. Vtg has been induced by administration of estradiol into S. marmoratus, and purified by gel filtration and ion-exchange chromatography from serum of the fish. After immunization of the purified Vtg into BALB/c mouse, hybridomas secreting anti-Vtg antibodies have been produced. The size of induced Vtg in the serum was about 440 kDa by gel filtration using Sepharose CL-6B. By SDS-PAGE analysis, the main band of Vtg, however, was at 175 kDa, and several minor bands have been detected with the main band. Eight different monoclonal antibodies have been produced from established hybridomas and the antibodies did not cross-react with sera from different species of fishes tested in this study except with that of Sebastes hubbsi. These results suggested that the monoclonal antibody of S28 and S15 can used as capture and tracer antibodies for ELISA and ICG assays. The detection systems developed in this study can be used as Bio-marker assays to check endocrine disrupting activity of various chemicals as well as to detect known endocrine disrupting chemicals contaminated in environment.

• The Journal of Pediatrics of Korean Medicine
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• v.32 no.4
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• pp.71-86
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• 2018

Objectives The purpose of this study is to investigate the effects of Gardenia jasminoides for. grandiflora extracts' (GAJ) anti-inflammatory effect on RBL-2H3 mast cells and OVA/alum-sensitized mice. Methods In this study, IL-4 and IL-13 production was measured via ELISA analysis, and mRNA expressions of GM-CSF, IL-4, IL-5, $TNF-{\alpha}$, IL-6 were analyzed by real-time PCR. In addition, MAPKs and $NF-{\kappa}B$ p65 transcription factors were examined using western blotting, and ELISA was used to understand IgE, IL-4, and IL-13 production in ovalbumin-allergic mice in in vitro study. Results As a result of this study, 1. GAJ were observed to suppress the mRNA expression of GM-CSF, IL-4, IL-13, IL-5, $TNF-{\alpha}$, IL-6 in comparison to PMA 50 ng/ml, ionomycin $0.5{\mu}M$ (PI) control group. 2. GAJ also inhibited the IL-4, IL-13 production in comparison to PI control group. 3. Western blot analysis showed decrease on the expression of mast-cell-specific transcription factors, including MAKPs (ERK, JNK, p38) and $NF-{\kappa}B$ p65. 4. Orally-administered GAJ group in OVA/alum induced Balb/c mice showed decreased level of OVA-specific IgE in the serum. This group also has shown decreased the level of IL-4, IL-13 in the splenocyte culture supernatant. Conclusions Obtained results suggest that GAJ may regulate the allergic inflammation by transcription factors MAKPs (ERK, JNK, p38) and $NF-{\kappa}B$ p65 causing inhibition of Th2 cytokines in mast cells and OVA/alum-sensitized mice.

• Journal of Life Science
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• v.32 no.12
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• pp.938-946
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• 2022

This study aims to evaluate the effects of antioxidant activities, protein and mRNA expressions of matrix metalloproteinase (MMP) -1 and procollagen type I C-peptide (PIP) in 70% ethanol extract from Hydnocarpus anthelmintica Pierre (HE). DPPH and ABTS⁺ radicals scavenging assays were measured for antioxidant activities and HE had 73.5% and 74.4% of scavenging activities at 1,000 ㎍/ml concentration, respectively. And we investigated the inhibition of collagenase by HE, and the result was a 78.8% inhibition effect on concentrations of 1,000 ㎍/ml. In addition, an MTT assay was performed to confirm the toxicity of the CCD-986sk fibroblasts to the HE, and as a result, the cell viability rate was about 91.7% at a concentration of 50 ㎍/ml or less, and subsequent cell experiments were performed at a concentration of 50 ㎍/ml or less. We treated the cells with UVB (20 mJ/cm²) for stimulation, treated HE at various concentrations, and performed ELISA tests and RT-PCR experiments. And HE increased the PIP and mRNA in a dose-dependent manner and showed an expression rate of about 64.2% and 83.4%, respectively, at a concentration of 50 ㎍/ml compared with Cont (50.3% and 45.8%, respectively). And HE suppressed the MMP-1 protein and mRNA in a dose-dependent manner and showed a low expression rate of about 48.7% and 35.9%, respectively, at a concentration of 50 ㎍/ml. These results can be applied to developing anti-wrinkle materials for functional food and cosmetics with HE.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.50 no.1
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• pp.67-75
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• 2024

Skin aging progresses due to external factors such as ultraviolet rays and infections. These factors cause skin fibroblasts to secrete proteolytic enzymes, matrix metalloproteinases (MMPs). MMPs induce the degradation of collagen located in the extracellular matrix, directly influencing aging. The stems of Akebia quinata Decaisne have been reported to have antioxidant and anti-inflammatory effects. However, the anti-aging effect of Akebia quinata Decaisne stem ethanol extract (AQSEE) is not known. Therefore, we studied the TNF-α-induced MMP-1 inhibitory effect in human fibroblasts. When the cell viability of AQSEE was confirmed through MTT asaay, it showed no toxicity up to 400 ㎍/mL. The inhibition of MMP-1 mRNA and protein secretion was confirmed through RT-qPCR and ELISA, and results showed a significant decrease at concentrations of 100, 200, 400 ㎍/mL. We also confirmed by Western blotting that phosphorylation of MAPKs signaling pathway and transcription factors was reduced. As a result, phosphorylation of p38, c-Jun, p65 was significantly decreased at all concentrations. DPPH and ABTS assays were performed to confirm the radical scavenging ability of AQSEE, and the results showed a significant decrease at all concentrations. The results of this study confirmed the MMP-1 inhibitory effect and radical scavenging ability, which suggests that it can be used as an anti-aging substance.