• IMMUNE NETWORK
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• 제12권1호
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• pp.33-39
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• 2012

Background: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). Methods: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reversetranscriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. Results: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. Conclusion: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

• Journal of Microbiology and Biotechnology
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• 제22권5호
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• pp.622-627
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• 2012

Mast cells and basophils are important effector cells in immunoglobulin-E (IgE)-mediated allergic reactions. Using the human basophilic KU812F cells, we assessed the inhibitory effects of 6-methoxyluteolin, isolated from Chrysanthemum zawadskii, in the $Fc{\varepsilon}RI$-mediated allergic reaction. We determined that 6-methoxyluteolin inhibited anti-$Fc{\varepsilon}RI$ ${\alpha}$ chain antibody (CRA-1)-induced histamine release, as well as elevation of intracellular calcium concentration $[Ca^{2+}]_i$ in a dose-dependent manner. Moreover, the inhibitory effects of 6-methoxyluteolin on the cell surface expression and the mRNA level of the $Fc{\varepsilon}RI$ ${\alpha}$ chain were determined by flow cytometric analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Therefore, these results show that 6-methoxyluteolin is a potent inhibitor of histamine release and calcium influx via down-regulation of the $Fc{\varepsilon}RI$ ${\alpha}$ chain.

• 대한화장품학회지
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• 제30권2호
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• pp.227-233
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• 2004

자외선이 피부에 지속적으로 조사되면 썬번, 염증, 광노화와 같은 다양한 부작용이 생기게 된다. 따라서 광손상에 대한 보호작용은 점점 중요성이 인식되고 있으며, 천연 식물추출물에서 자외선에 대한 광보호효과가 있는 안전하고 효과적인 물질개발에 대한 연구가 진행되고 있다. 본 연구는 자외선에 의해 유도된 피부손상에 대한 보호효과가 우수한 천연식물추출물에 대한 것이다. 향나무, 능소화, 비자추출물에서 자외선에 의해 생성되는 라디칼에 대한 항산화효과, MMP 발현 및 활성 저해, 염증관련 사이토카인인 인터루킨 1알파, 6, 프로스타글란딘 E$_2$ 생성저해효과 등으로 피부세포보호 효과를 연구하였다. 실험결과 천연추출물 중 향나무추출물과 능소화추출물이 프리라디칼 및 슈퍼옥사이드 라디칼 소거효과가 우수하게 나타났으며 피부 콜라겐과 같은 메트릭스를 분해하는 효소인 MMP-1의 활성 및 발현 저해효과는 섬유아세포에서 UVA 조사에 의한 실험에서 우수하게 나타났으며, 피부세포 배양액에 대해 zymography를 실시하여 활성이 감소됨을 확인하였다. 피부 각질형성세포에서 자외선에 의해 유도된 염증관련 사이토카인인 인터루킨 6의 발현량 실험에서도 무처리군에 비해 향나무추출물이 인터루킨 6을 30% 정도 저해효과가 나타났으며, 염증반응 중 cyclooxygenase(COX)에 의한 경로에서 생성되는 프로스타글란딘 E$_2$의 생성량도 감소시켰다. 사람피부에서 SLS (0.5%) 첩포로 유발된 자극성 피부염의 항염증 효과 평가 시 SLS에 의해 유발되는 자극정도가 피검자의 피부상태에 따라 다양하게 나타났으며, 향나무 추출물 함유 에멀젼 제품 도포 실험에서는 피부 홍반 완화 효과와 피부장벽 회복효과가 우수하게 나타났다. 이상의 결과로 향나무추출물은 자외선 조사 및 SLS에 의한 피부손상에 대한 피부세포보호작용이 우수하여 광노화에 대응하는 자극완화 소재로서의 화장품 응용 가능성을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7857-7861
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• 2014

Natural killer (NK) cells play an important role in anti-tumor immunity. Interleukin (IL)-18 is an immunoregulatory cytokine that induces potent NK cell-dependent anti-tumor responses when administrated with other cytokines. In this study, we explored the effects of combining IL-18 and IL-2 on NK cytotoxicity as well as expression levels of the NK cell receptor NKG2D in vitro. Freshly isolated PBMCs were incubated for 48 h with IL-18 and IL-2, then CD107a expression on $CD3^-CD56^+$ NK cells was determined by three-colour flow cytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human colon carcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on NK cells. The combined use of IL-18 and IL-2 significantly increased CD107a expression on NK cells compared with using IL-18 or IL-2 alone, suggesting that the combination of these two cytokines exerted synergistic enhancement of NK cytotoxicity. IL-18 also enhanced NKG2D expression on NK cells when administered with IL-2. In addition, blockade of NKG2D signaling with NKG2D-blocking antibody attenuated the up-regulatory effect of combining IL-18 and IL-2 on NK cytolysis. Our data revealed that IL-18 synergized with IL-2 to dramatically enhance the cytolytic activity of human NK cells in a NKG2D-dependent manner. The results appear encouraging for the use of combined IL-18 and IL-2 in tumor immunotherapy.

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• BMB Reports
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• 제35권5호
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• pp.524-531
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• 2002

Thrombotic thrombocytopenic purpura (TTP) is characterized by widespread platelet thrombi in arterioles and capillaries. Unusually large or multimeric von Willebrand factor, as well as one or ore platelet-agglutinating factors, have been implicated in the pathogenesis of TTP. But, the actual mechanisms of platelet agglutination have not been satisfactorily explained. Recent studies suggested the 37-kDa platelet-agglutinating protein (PAP) p37 to be partially responsible for the formation of platelet thrombi in patients with TTP. We studied mobility in SDS-PAGE, the sequence of N-terminal amino acid residues, DNA and antigenic characteristics of PAP p37, which might be related to the pathogenesis of TTP. PAP p37 was purified from the plasma of a 31-year-old male Korean patient with acute TTP. The findings are as follows: (1) We compared PAP p37 with thrombin through the use of SDS-PAGE, either with or without $\beta$-mercaptoethanol. PAP p37 did not appear to be cleaved between the A- and B-chains of prethrombin 2. However, thrombin did cleave between those of prethrombin 2, but linked with disulfide bridge. (2) N-terminal 21 amino acid sequence of PAP p37 was T-F-G-S-G-E-A-D-X-G-L-R-P-L-F-E-K-K-S-L-E. It appeared to be identical to that of 285-305 amino acid residues of human prothrombin (prethrombin 2). (3) No prothrombin gene DNA mutation was revealed. (4). The antigenicity of PAP p37 was similar to thrombin, which was a result of the competitive binding against the anti-thrombin antibody. With these results, we conclude that PAP p37 has similar characteristics to prethrombin2.

• Childhood Kidney Diseases
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• 제9권2호
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• pp.143-148
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• 2005

목 적 : 소아 신증후군에서 알부민을 비롯한 단백들과 IgG 치의 감소가 관찰된다. 신증후군에서 관찰되는 IgG 감소에서 IgG 아군간에 차이가 있는 지와 B형 간염 및 홍역 항체의 양성률이 대조군과 차이가 있는지를 알아보았다. 방 법 : 가톨릭대학교 대전성모병원 소아과에 신증후군으로 입원하였던 21명과 같은 연령대의 건강한 환아 25명을 대상으로, 혈청에서 IgG, IgM, IgA, IgE 및 IgG subclasses(IgG1, IgG2, IgG3 및 IgG4), B형 간염 표면 항체(anti-hepatitis B suface IgG, anti-HBs IgG) 및 항홍역 IgG 항체(anti-measles IgG)를 측정하였다. 결 과 : 신증후군 환아들의 평균 연령 $6.9{\pm}3.0$세로, 면역글로불린 평균값은 IgG $390{\pm}187\;mg/dL$, IgG1 $287{\pm}120\;mg/dL$를 보였다. 대조군의 평균 연령은 $7.5{\pm}3.4$세로, IgG $1,025{\pm}284\;mg/dL$, IgG1 $785{\pm}19\;mg/dL$이었다. IgE 값에서 250 IU/mL 이상을 보인 경우는 신증후군에서 11명(52.4$\%$), 대조군에서 7명(28$\%$)이 있었다(.P=0.01). 신증후군 환아에서 IgG 및 IgG 아군 모두에서 유의한 감소를 보였으며(P<0.001), IgM 값은 증가($251{\pm}183\;mg/dL\;vs.\;153{\pm}55\;mg/dL$, P=0.02)를 보였으나 IgA 값은 차이를 보이지 않았다. 한편 Anti-HBs의 양성률은 신증후군 환아군에서 42.9$\%$(21명 중 9명), 대조군에서 52$\%$ (13/25명)를, 항홍역 항체 양성률은 각각 76$\%$ (16/21명)와 92$\%$ (23/25명)를 보였으나 통계학적으로 두 군간에 차이는 없었다. 결 론 : 소아 신증후군 환아에서 IgG 및 모든 IgG 아군의 감소를 보였다. 예방접종에 의한 B형 간염 및 홍역 항체 양성률의 감소가 관찰되었으나 통계학적인 차이는 보이지 않았다. IgG 감소의 기전에 대한 후속 연구가 필요할 것으로 보인다.

• Parasites, Hosts and Diseases
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• 제27권2호
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• pp.109-118
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• 1989

폐흡충 감염시 숙주 말초혈액에는 많은 항체가 생성되므로 각종 면역학적 진단에 이용되 고 있다. 그러나 이들 항체가 폐흡충 충체 구성 요소 중 주로 어느 기관(또는 구조물)에 대한 항체 인지에 대해서는 별로 연구된 바 없었다. 이 연구는 면역 조직화학적 방법을 이용하여 폐흡충 충체 의 부위별 항원성을 파악하기 위한 것으로 충체 절편 표본에서 흡반, 표피, 피극, 난황선, 장, 자성 및 웅성 생식기, 충란 등의 항원성을 비교 관찰한 것이다. 충체 표본으로는 감염 11∼20주 된 고양이의 폐조직에서 충낭(WOrm capsule)을 적출하여 포르말 린 고정 및 파라핀 포매한 것을 4㎛ 두께로 영아 사용하였고, 항혈청(1차 항체)으로 감염 11∼20 주 된 고양이 혈청을, 2차 항체로 peroxidase-conjugated goat anti-cat IgG를 사용하는 간접 면역 대소 염색법(indirect immunoperoxidase staining technique)을 이용하였고, 진한 황색 또는 황갈색으로 염색되면 양성으로 판정하였다. 항체의 희석 농도는 1차 항체 1 : 500~1 : 2,000, 2차 항체 1 : 200~1 : 500으로 하였고 10회 이상 반복 염색하였다. 실험 결과 장 상피의 표면(intestinal epithelial border), 장 내용물, 난황선(vitelline glands) 및 충낭 내의 충란(eggs in worm capsule) 등이 강한 양성 반응을 보였고 자궁 내 충란 및 충체 실질 조직 중 일부도 약하지만 양성 반응을 보였다. 한편 흡반(suckers), 표피(tegument), 피극(spines), 표피하세포(subtegumental cells), 장 상피세포의 세포질, 웅성 생식기관 및 난소 등은 음성 반응 을 나타내었다. 항원성의 강도를 순서대로 나열해보면 장 상피의 표면, 장 내용물, 충낭 내의 충란, 난황선, 자궁 내 충란, 충체 실질조직의 순이었다. 항원성이 강한 장 상피층 및 장 내용물은 1차 항체 1 : 4,000(2차 항체 1 : 200)에서도 양성 반응을 보였으나 충체 실질조직 중 일부는 1차 항체 1 : 500의 고농도(2차 항체 1 : 200)에서만 양성 반응을 보였다. 이상의 결과를 종합해 볼 때, 폐흡충 감염시 나타나는 혈청의 항체 반응은 충체의 배설물과 충 낭 주위에 산출된 충란에 의해 가장 강력히 유발되는 것이 아닌가 생각되며 이들이 충낭을 벗어나 숙주 조직으로 총수되는 가장 중요한 항원성 물질이 아닌가 추측된다.

• 한방안이비인후피부과학회지
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• 제19권2호
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• pp.1-18
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• 2006

Objective : Although management of asthma has become increasingly effective, its cure remains elusive, necessitating a new modality to prevent or eliminate causes triggering clinical progress. Based in the clinical experiences, a novel decoration Cheongsangbiyeum (CSB), has been developed to treat asthma, which consists of Polyporus, Semen Myristicae, Pericarpium citri Reticulatae, Rhizoma Cimicifugae, Cortex Albizziae, Fructus Rubi, Rhizoma Zedoariae, and Rhizoma Rhei. In the current study, its anti-asthmatic efficacy was evaluated using a mouse model of asthma. Methods : Experimental allergic asthma was induced by repeated intraperitioneal sensitization and intranasal challenge of ovalbumin (OVA). Water extract of CSB (1 mg/mouse/day) was administrated orally whereas control mice on given with identical volume of phosphate-buffered saline (PBS) for 5 days during the course of antigen challenge. When airway hyperreactivity(AHR) measured by ${\bata}-methacoline-induced$ airflow obstruction was compared, AHR of CSB-treated mice was significantly lower than those of control mice, indicating that CM extract can attenuate an asthmatic symptom. Airway recruitment of leukocytes and eosinophils was also markedly reduced by CSB treatment suggesting that oral treatment of CSB can alleviate the airway inflammation. For a better understanding of possible mechanisms underlying anti-asthmatic effet of CSB, cytokine (IL-4, IL-5, IL-13 and $IFN{\gamma}$ levels in bronchoalveola lavage fluid (BALF) and lung tissues were determined. Results : The results showed that cytokine levels were significantly lowered by CSB treatment. Additionally, number of draining lymph node cells was significantly lower than those of control mice. These data indicate that CSB suppress in vivo allergen-specific response. However, notably, levels of type 2 cytokines such as IL-5 and IL-13 were more profoundly influenced. Moreover, in vitro OVA-specific proliferative response and type 2 cytokine (IL-4, IL-5 and IL-13) production lymph node cells was markedly decreased in CSB-treated mice, whereas their $IFN{\gamma}$ production was not significantly altered Thrse data clearly showed a preferential inhibition of type 2 T cell (Th2) response by CSB treatment. This finding was also supported by serum antibody data showing that levels of OVA-specific type 2 antibodies, IgE and IgG1, in CSB-treated mice were significantly lower than in control mice, while type 1 antibody, IgG2a level m rather higher than controls, although the difference was in significant. Conclusions : In conclusion, oral administration of CSB attenuates asthmatic manifestations including AHR ad airway recruitment of eosinophils in a mouse model which possibly results from selective inhibition of Th2 cell response to allergen. Our data suggest a potential clinical application of CSB for control of allergic asthma.

• Journal of Pharmaceutical Investigation
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• 제29권2호
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• pp.87-92
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• 1999

Drug delivery to the brain may be achieved by producing chimeric peptide, attaching the drug to protein 'vectors' which are transported into the brain from the blood by a receptor-mediated transcytosis through the blood-brain barrier (BBB). Since the BBB expresses high concentrations of transferrin receptor, and it was reported that anti-transferrin receptor mouse monoclonal antibody (OX26) undergoes transcytosis through the BBB, it is logical to assume that a drug delivery system via transferrin receptor-mediated transcytosis is a promising strategy. In the present study, therefore, we tested feasibility of several OX26 based vectors for the brain delivery of a model drug. Avidin-based delivery vectors such as OX26-streptavidin (OX26-SA), OX26-neutralite avidin (OX26-NLA) were chemically synthesized vectors and OX26 immunoglobulin G 3 type $C_{H}3$ fusion avidin $(OX26\;IgG3C_H3-AV)$ was genetically engineered. To improve the efficiency of producing chimeric peptide, we used avidin-biotin technology. Pharmacokinetics of $[^3H]biotin$ bound to OX26-SA, OX26-NLA and $OX26\;IgG3C_H3-AV$ was determined by intravenous injection technique, and their stabilities in plasma were analyzed using HPLC. The brain delivery of $[^3H]biotin$ bound to OX26-SA, OX26-NLA and OX26\;$IgG3C_{H}3-AV$ (expressed as %ID/g brain) was $0.22{\pm}0.01$, $0.18{\pm}0.01$ and $0.25{\pm}0.09$, respectively. The areas under the plasma concentration versus time curve (AUC) for OX26-SA, OX26-NLA, $OX26\;IgG3C_H3-AV$ from time zero to 60 min were $209{\pm}10$, $195{\pm}9$, $134{\pm}29\;%ID\;min/ml$ respectively and their total clearances $(CL_{tot})$ were $1.00{\pm}0.09$, $1.08{\pm}0.07$ and $1.54{\pm}0.29\;ml/min/kg$, espectively. These results showed that these vectors possess preferable pharmaceutical (e.g., resonable stability) and pharmacokinetics (e.g., significant brain uptake and enhanced AUC) for brain delivery. Therefore, these vectors may be broadly useful in the brain delivery of drugs that are not transported into the brain to a significant extent.