• IMMUNE NETWORK
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• 제16권5호
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• pp.296-304
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• 2016

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation.

• Animal cells and systems
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• 제15권4호
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• pp.310-314
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• 2011

The Banna miniature pig (BNMP) is a representative miniature pig breed in China. Even though BNMP dwarfism is obvious, its underlying causative mutations remain unknown. In this study, the BNMP and Large White pig (LWP) serum growth hormone (GH) and insulin-like growth factor (IGF-1) levels were detected by ELISA and compared. BNMP serum IGF-1 levels were significantly lower than LWP levels (P<0.05). The miniature condition may arise from mutations in the GH and GH receptor (GHR) genes. Therefore, GH and GHR cDNA from the BNMP were cloned into a pMD18-T vector by RT-PCR using the total RNA obtained from the BNMP's pituitary and liver tissues. Sequencing results indicated that the open reading frame of the BNMP GH gene is composed of a 26-residue signal peptide and a 191-residue mature peptide. The coding sequence of the BNMP GHR gene contained 639 amino acids, including a signal peptide that is 18 amino acids long. Two amino acid substitutions, A09V and R22Q, were found in the signal peptide of the GH gene. Additionally, the S104P mutation was found in the BNMP's mature GH protein. Four mutations in the cytoplasmic domain of GHR may influence the downstream signal transduction of GHR, which needs further experimental evidence.

• IMMUNE NETWORK
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• 제2권3호
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• pp.133-136
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• 2002

Background: The costimulatory molecule 4-1BB, a member of nerve growth factor receptor/tumor necrosis factor (NGFR/TNFR) super family, is involved in cell survival and death. Methods: In this study, female C57BL/6 ($H-2^b$) mice were used as a recipient, and DBA/2 ($H-2^d$) as a donor to assess a mixed lymphocyte reaction (MLR) and CTL response in vitro, and skin graft survival. IL-2, IFN level was measured by ELISA. Results: Mixed lymphocyte reaction (MLR) analysis showed that 4-1BB-deficient responder cells showed enhanced cellular proliferation over littermate controls. In contrast, IL-2 production was diminished only in 4-1BB knockout cultures. The IFN expression, on the other hand, was comparable between the groups. When female C57BL/6 ($H-2^b$) mice were grafted with the trunk skin of DBA/2 ($H-2^d$) mice, the in vivo tissue destruction of 4-1BB-deficient mice was not distinct from the normal littermates. Conclusion: These data suggest that 4-1BB is critical for the induction of alloreactive responses in vitro but 4-1BB alone could not change the course of skin rejection in vivo.

• 생약학회지
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• 제44권2호
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• pp.104-109
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• 2013

Hesperidin (HES), a flavonone glycoside isolated from the citrus fruits such as lemons and oranges, has been reported to have many biological properties including antiinflammatory, antioxidant, and antiallergy activities. In this study, we focused on the action of HES modulating Th2-associated cytokines such as IL-4 and IL-13 expression in PMA/ionomycin (PI)-stimulated rat basophilic leukemia (RBL-2H3) cells. The production of IL-4 and IL-13 was quantified by ELISA and the mRNA expression was detected by using RT-PCR assay. In addition, western blot analysis was performed to determine the transcription factors involved in the cytokine expression. We found that HES significantly decreased PI-induced IL-4 and IL-13 productions and also decreased the level of mRNA in a dose-dependent manner. Furthermore, western blot analysis of the transcription factors implied that HES down-regulated the protein level of c-Jun and c-Fos, which are the activating protein 1 (AP-1) family and nuclear factor-kappaB (NF-${\kappa}B$) characterized as a transcription factors related to the Th2-associated cytokine expression. Taken together, our data showed that the action of HES responsible for antiallergy activities is based on suppression of Th2-associated cytokines through inhibition of AP-1 and NF-${\kappa}B$ transcription factors.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.718-724
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• 2017

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and half-life. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

• 한국환경농학회지
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• 제13권3호
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• pp.262-270
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• 1994

Endosulfan의 잔류분석(殘留分析)을 위한 RIA(radioimmuno assay)는 endosulfan alcohol(EA)-BSA conjugate를 항원(抗原)으로 하여 집토끼에서 면역항체(免疫抗體)를 생산(生産)하였다. 생산(生産)된 면역항체(免疫抗體)의 역가(力價)는 1 : 32,000 으로 endosulfan과 EA 이외(以外)의 유사화합물(類似化合物)에서는 거의 반응성(反應性)을 나타내지 않았으며 RIA를 위한 최적(最適) 희석배수(稀釋倍數)는 1 : 2,000 으로 나타났다. RIA의 최적(最適) 배양온도(培養溫度)는 $4\;^{\circ}C$ 이었으며 endosulfan의 검출범위(檢出範圍)는 $1\;ng-20\;{\mu}g$ 이었고 최소검출량(最小檢出量)은 1 ng 이었다. RIA 기법(技法)을 이용(利用)하여 잉어의 각(各) 조직(組織)과 공시수(供試水)에서 실시한 회수율(回收率) 시험(試驗) 결과(結果)는 GLC/ECD나 ELISA를 이용(利用)한 회수율(回收率)보다 우수하게 나타났다. 시료(試料)에서 RIA에 의한 endosulfan의 검출한계(檢出限界)는 0.1 ppb 였다.

• Journal of Microbiology
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• 제40권4호
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• pp.313-318
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• 2002

To perform the prophylactic study of a vaccine derived from human papillomavirus (HPV) using Balb/c mice, we produced virus like particles consisting of HPV capsid protein L1 which has been reported to induce significant humoral and cellular immunity using various animal model systems. In order to produce HPV16 VLPs, the cDNA of L1 capsid protein in HPV type 16, obtained by polymerase chain reaction, was inserted into yeast expression vector, YEG$\alpha$-HIR525 under the control of GAL10 promoter. The transformation of YEG$\alpha$-HPV16 L1 was performed into the yeast Saccharomyces cerevisiae Y2805 by the lithium acetate method and the yeast clone expressing the highest level of L1 capsid protein of human papillomavirus type 16 was selected by Western blot analysis using anti-HPV16 L1 antibody. The purification of HPV16 VLP has been performed by the ultracentrifugation and gel-filtration methods. To validate the vaccine efficacy of the purified HPV16 VLPs and investigate the properties of HPV16 VLPs to induce humoral immunity, ELISA assay was performed. A significantly increased production of anti-HPV16 VLP antibodies was observed in sera from immunized mice. The neutralization activity of antibodies in the sera from the vaccinated mice was demonstrated by a rapid and simple assay to detect hemagglutihation inhibition activity.

• 대한한방소아과학회지
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• 제26권1호
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• pp.1-15
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• 2012

$CheongGiGeoYangTang$ has been used for anti-allergenic purpose. However there was no experimental study about its effect. Therefore, this study was designed to investigate the anti-allergenic effect of $CheongGiGeoYangTang$. Methods Modifiability of RBL-2H3 mast cells' IL-4, IL-13 was analyzed by qRT-PCR and ELISA. Also, the suppressive effect of GATA-1, GATA-2, NF-AT1, NF-AT2, AP-1 and NF-${\kappa}B$ p65 transcription factors was observed by western blotting. OVA-specific IgE, IL-4 and IL-13 production in ovalbumin allergy model was examined as well. Results It was showed that the RBL-2H3 mast cells treated with $CheongGiGeoYangTang$ extract(CGGYT) was significantly suppressed mRNA expression, production of IL-4 and IL-13, and prominently inhibited the expression of transcription factors including GATA-1, GATA-2, NFAT-1, NFAT-2, c-Fos and NF-${\kappa}B$ p65 but not c-Jun. The administration of CGGYT was suppressed the amount of OVA-specific IgE, IL-4 and IL-13 in OVA/alum-sensitized mice. Conclusions We considered CGGYT would regulate the allergic inflammation as inhibition of IL-4 and IL-13 production in activated mast cells and Th2 cells.

• 동의생리병리학회지
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• 제18권2호
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• pp.580-585
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• 2004

BJYGC is often clinically used as a treatment of allergic rhinitis. This study was aimed to find out the effect BJYGC would have on the helper T cell, and how it can promote the subsets of helper T cells to regain their balance that they lost due to immunological diseases. Splenocytes were prepared from BALB/c mice was cultured without stimulation in the presence of BJYGC for 48 hr. The viability of CD4 T cells from Balb/c mouse were measured at various concentrations of BJYGC using the MTS assay. It was somewhat increased up to concentration of 400 ㎍/ml, but did not show any significant difference. Proliferation was measured using the MTS assay, CD4 Th cells were stimulated with anti-CD3/28 in the presence of BJYGC for 48 hr. As evidence for rapid T cell activation, CD25 expression by flow cytometry was evaluated at 10, 50, 100 and 200 ㎍/㎖ of BJYGC. Th cell differentiation experiments were performed to examine whether BJYGC can affect the Th polarization process. CD4 T cells were activated in culture under neutral, Th1-polarized or Th2-polarized conditions in the presence of BJYGC at 10, 100 and 200 ㎍/㎖. Cytokine production was measured by ELISA. This experiment proved that BJYGC could inhibit the secretion of both IL-4 and IFN-γ in neutral condition and polarized condition, too. Considering that BJYGC shows an excellent effect on treating allergies, the author can conclude that its pharmacological action may be associated with decreased IL-4 and, it may also regulate IFN-γ depending the host's need. Also, it was discovered that Th1 cell was pathologic in chronic inflammatory tissue specific diseases, such as insulin dependent diabetes mellitus, multiple sclerosis, RA, and uveitis. We are counting on the BJYGC to be able to control the tendency of Th1 cell predominancy in an immune reaction.

• Reproductive and Developmental Biology
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• 제38권3호
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• pp.99-105
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• 2014

In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhibition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.