• 대한미생물학회지
• /
• 제21권1호
• /
• pp.151-161
• /
• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.

• Clinical and Experimental Pediatrics
• /
• 제45권7호
• /
• pp.875-883
• /
• 2002

목 적 : 기관지 천식은 Th2 반응 우세로 인한 기도의 호산구증과 IgE 증가를 특징으로 하는 질환이다. 박테리아 DNA 혹은 합성 ODNs인 unmethylated CpG motifs(CpG)은 Th1 사이토카인인 $INF-{\gamma}$와 IL-12 유도와 Th2 사이토카인을 하강 조절하여 기도 내의 호산구증 및 전신적 IgE 생성을 억제하며, 메타콜린 흡입에 의한 기관지 과민성을 방지한다. 저자들은 기도 염증을 유발한 생쥐의 비강내에 합성 CpG ODNs의 점적 투여로 기도의 호산구증과 그 외 면역학적 기전에 대한 영향을 연구하고자 한다. 방 법 : BALB/c 생쥐에 주당 2번씩 면역보강제 없이 OVA를 $10{\mu}g$을 피하 주사하여 감작 시켰다. 10주째에 $50{\mu}g$ OVA를 백서의 비강내로 주입하여 기도 염증을 유도한 후, 4주 뒤인 14주째 다시 생쥐의 비강내에 OVA＋CpG ODNs과 OVA를 점적 흡입 주입하였다. 마지막 항원 유발(14주) 후 4일째에 기관지 폐포 세척액을 추출하여 세포를 염색한 후 호산구를 측정하였고 동시에 비장 세포를 배양하여 사이토카인 측정을 하였다. 비장 세포에서 OVA에 의해 생성된 사이토카인 즉 Th1($INF-{\gamma}$, IL-12), Th2(IL-4, IL-5, IL-13) 사이토카인을 ELISA에 의해 측정하였다. 혈청 항원 특이 IgE, IgG2a 또한 ELISA에 의해 측정하였다. 결 과 : 1) 기관지폐포 세척액의 호중구수 : CpG ODNs을 투여한 군이 7.6[5.8-9.4]%, CpG ODNs을 투여하지 않은 군이 42.0[38.2-44.6]%로 기도 염증 세포내의 호산구증을 현저히 억제시켰다(P<0.01). 2) 혈청 OVA-specific IgE와 IgG2a : CpG ODNs으로 투여한 군과 투여하지 않은 군에서 측정한 혈청 OVA-specific IgE와 IgG2a는 음성 대조군에 비해 현저히 증가하였고, CpG ODNs을 비강내 투여 후 4일째 측정한 혈청 OVA-specific IgE와 IgG2a 수치는 CpG ODNs 투여군과 비투여군 사이에 유의한 차이가 없었다. 3) 사이토카인 분석 : CpG ODNs으로 투여한 군은 투여하지 않은 군에 비해 IL-4, IL-5, IL-13의 생성이 감소하였고 IL-12 생성은 증가하였으나, $INF-{\gamma}$의 상향조절은 보여주지 못하였다. 결 론: CpG ODNs의 백신 접종은 면역반응을 조절하여 기도 염증이 형성된 생쥐에서 기도 호산구증을 감소시키는데 매우 효과적이며 천식 치료에 유용할 수 있을 것으로 사료된다.

• 대한한방소아과학회지
• /
• 제29권4호
• /
• pp.39-66
• /
• 2015

Objectives The purpose of this study is to investigate the effects of Sesamum indicum extracted (SEI) on atopic dermatitis in an in-vitro and in-vivo experiment using a MC/9 murine mast cells and a NC/Nga mouse. Methods In-vitro experiment, IL-4, IL-5, IL-6, IL-13, TNF-${\alpha}$ and GM-CSF mRNA expression were evaluated by Real-time PCR, IL-13, MIP-$1{\alpha}$ production by ELISA and manifestations of NFAT-1, NFAT-2, c-jun, c-fos, NF-${\kappa}B$ p65 transcription factors by western blotting. In-vivo experiment, we measured WBC, Eosinophil, Neutrophil, and serum IL-5, IL-13 in NC/Nga atopic dermatitis mouse, IL-5, IL-13, IFN-${\gamma}$, IL-4 in the spleenocyte culture supernatant by ELISA, the absolute cell numbers of CD4+, CD8+, +Gr-1+CD11b, B220+CD23+ in the axillary lymph node (ALN), peripheral blood mononuclear cells (PBMCs) and dorsal skin tissue, IL-5, IL-13 by Real-time PCR, the distribution of tissue inflammation and cellular infiltration by H&E and toluidine blue. Results SEI decreased IL-4, IL-5, IL-6, IL-13, GM-CSF, TNF-${\alpha}$ mRNA expression, IL-13, MIP-$1{\alpha}$ production and the expression of transcription factors including NFAT-1, c-jun, NF-${\kappa}B$ p65 in MC/9 murine mast cells. SEI orally administration decreased cell number of WBC, Eosinophil, the level of serum IgE, total cell number of ALN and dorsal skin tissue, absolute cell number of CD4+, CD8+, B220+CD23+ in the ALN. SEI orally administration also increased absolute cell number of CD8+/CD3+ and decreased Gr-1+/CD11b+ in PBMCs, decreased CD4+ in dorsal skin tissue, inhibited IL-5, IL-13 mRNA expression. Infiltration levels of inflammatory immune cells, mast cells and thickness of epidermis decreased in dorsal skin tissue. Conclusions SEI can regulate allergic inflammatory response suppressed the gene expression and production of cytokines that mediate allergic reactions, and will be able to be effectively utilized in the treatment of atopic dermatitis future.

• 한국환경농학회지
• /
• 제32권3호
• /
• pp.231-239
• /
• 2013

본 연구는 병저항성 OsCK1 유전자와 제초제저항성 PAT 유전자가 도입된 병저항성 GM벼에서 도입 유전자의 발현 검증과 수서환경의 비표적 생물체인 잉어와 미꾸리에 미치는 영향을 확인하기 위해 수행되었다. 병저항성 GM벼에 도입된 OsCK1와 PAT 유전자의 PCR 분석 결과, 병저항성 GM벼에서만 특이적인 밴드가 검출되었으며, PAT 단백질 발현량을 ELISA 분석한 결과, 병저항성 GM벼에서만 $45.44{\pm}2.23{\mu}g/g$ 수준으로 검출되었고, 모품종인 낙동벼에서는 검출되지 않았다. 병저항성 GM벼와 낙동벼의 미꾸리(Misgurnus anguillicaudatus)와 잉어(Cyprinus carpio)에 대한 급성독성시험을 실시한 결과, 48시간 및 96시간-$LC_{50}$ 은 5,000 mg/L 이상으로 나타났다. 48시간 및 96시간 무영향농도 NOEC)는 5,000 mg/L이었다. 급성독성 시험기간 중 병저항성 GM벼와 낙동벼간의 pH, DO, 수온, 체중 및 전장에 대한 유의적인 결과는 나타나지 않았다.

• 생태와환경
• /
• 제38권2호통권112호
• /
• pp.237-248
• /
• 2005

서울 경기지역 공원 연못 및 한강 수계내의 수중 조류독소 Microcystin (MC)의 분포를 파악하기 위하여, 수도권 공원 연못 7개소, 한강수계 4개소, 경안천 1개소, 양수리 육각지 1개소, 대조군 2개소를 포함 총 15개 지점을 대상으로 강우가 적은 저온기 동안, 3차례에 걸쳐 수중내 환경요인, 조류 현존량, 조류 독소량을 조사하였다. 조류독소는 유도체중 가장 독성이 강한 Microcystin-LR (MCLR)만을 대상으로 하였으며 ELISA법으로 분석하였다. 조사기간 동안 수온($0.4\;{\sim}\;21.9^{\circ}C$)을 비롯한 대부분의 환경요인들은 계절성과 지역성이 뚜렷하였다. 조류 현존량은 수온 변화에 민감하였고 (r = 0.307), Microcystis aeruginosa를 포함하여 남조류는 경복궁 경회루, 석촌호수. 양수리 육각지 등에서 우점하였다. 조류독소 MCLR (측정한계 $0.05\;{\mu}g\;L^{-1}$) 는 남조 Microcystis aeruginosa출현이 높았던 지점에서 높게 검출되었으며, 조체성과 용존성을 합한 총 MCLR은 1차조사시 경복궁 경회루 ($1.39\;{\mu}g\;L^{-1}$), 석촌호수($0.55\;{\mu}g\;L^{-1}$), 양수리 육각지 ($0.59\;{\mu}g\;L^{-1}$)에서 각각 높게 나타났으나,2, 3차 조사에서는 양수리 육각지에서만 검출되었다. 남조류 총현존량은 조류 현존량 (r = 0.066)이나 엽록소 a량(r = -0.058)과는 낮은 관계를보였으며, M. aeruginosa 현존량(r = 0.766)과는 매우 높은 관계를 나타냈다. 조류독소 MCLR은 남조 M. aeruginosa 현존량이 높았던 지점에서 높게 검출되었다(조체성: r = 0.526, 용존성: r = 0.433).따라서, 서울, 경기지역 공원 연못이나 한강수계의 조류독소는 주로 정체성 연못에서 남조 Microcystis aeruginosa에 의해 형성되지만, 여가활동이나 상수원 공급에는 위험성이 낮은 것으로 판단되었다.

• 동의생리병리학회지
• /
• 제19권5호
• /
• pp.1281-1291
• /
• 2005

Chungpae-tang is suggested to have the antitumor activity on lung cancer. This study was peformed to investigate apoptotic effect in vitro and antitumor effect and immune response after injection of B16-F10 melanoma cells and Chungpae-tang into a tail vein of C57BL/6 mice and administratition of Chungpae-tang in A549 human lung cancer cell line in vivo, respectively. Experimental studies were obtained by measuring the median survival time and cytokine expression through RT-PCR, and ELISA assay. The results were summarized as follows: 5 mg/ml of Chungpae-tang causing DNA fragmentation, caspase-3 enzyme activation, PARP fragmentation, and cytochrome c release, suggested that Chungpae-tang has in vitro apoptotic effect in A549 human lung cancer cell line in the apoptosis-induced experiment. The median survival time of the Chungpae-tang treated group was 21 days and that of control group was 22 days, suggesting that the median survival time between the Chungpae-tang treated group and the control group was not significant. Cytokine expression between the Chungpae-fang treated group and the control group was noticeable, but was not significant in the RT-PCR. In the ELISA assay, IL-2 productivity in the Chungpae-tang treated group was to increase more than that in the normal group (p<0.05) and was no significant between the Chungpae-tang treated group and the control group. $INF-\gamma$ productivity of the control group decreased more than that of the normal group (p<0.05) and that of the Chungpae-tang-treated group increased more than that of the control group (p<0.05). IL-12 productivity of the control group increased more than that of the normal group (p<0.05) and that of the Chungpae-tang-treated group decreased more than that of the control group (p<0.05) and the normal group. IL-4 productivity of the Chungpae-tang-treated group increased more than that of the normal group and the control group (p<0.05). IL-10 productivity of the Chungpae-tang-treated group increased more than that of the normal group and the control group (p<0.05). Accordingly the results show Chungpae-tang could induce apoptosis in A549 human lung cancer cell line and bring to antitumor effect and immune response against injection of B16-F10 melanoma cells into a tail vein of C57BL/6 mice but it needs more research on the precise mechanism of such effects.

• 한국식품영양학회지
• /
• 제28권6호
• /
• pp.1090-1097
• /
• 2015

겨우살이는 전통적으로 항암활성이 있는 약용식물의 하나로 알려져 왔고, 렉틴은 세포독성 및 면역자극 자극 활성을 가지는 대표성분으로 인정되고 있다. 한국산 겨우살이에 함유되는 렉틴은 유럽산의 그것과는 달리 galactose와 N-acetylgalactosamine(GalNAc) 특이성을 동시에 가지는 렉틴 성분인 KML인 것으로 나타났다. Sandwich ELISA법을 이용하여 각기 다른 종류의 숙주나무에서 유래된 5종의 겨우살이로부터 렉틴 함량을 비교한 결과, 숙주나무별 차이가 인정되어 밤나무 겨우살이는 참나무 겨우살이에 비하여 약 10배 많은 렉틴을 함유하고 있었다. L5178Y-ML25 lymphoma 세포에 약 90%의 세포독성을 나타내는 농도의 KML과 한국산 참나무 유래 겨우살이 추출물인 KM-100에 두 종류의 단일클론 항체(9H7-10 and 8B11-2C5)를 동시처리한 후 세포독성 중화효과를 조사한 결과, KML의 경우 약 10%, KM-110의 경우 약 30%의 세포독성을 보였다. 이러한 결과는 겨우살이에서 렉틴 외에도 세포독성을 가지는 다른 성분이 존재할 것으로 사료되었다. RAW 264.7 대식 세포주에 KM-110과 KM-110으로부터 렉틴이 제거된 분획인 LFKM-110을 자극시킨 결과, LFKM-110에서 $TNF-{\alpha}$ 및 IL-6와 같은 cytokine의 생산을 증진시키는 결과를 보였다. 따라서 KM-110에서 면역 세포를 자극하는 다른 성분의 존재하고 있음을 강하게 제시되었다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권22호
• /
• pp.9713-9717
• /
• 2014

Background: Colon cancer (CRC) is perhaps the second most common cause of cancer mortality. This study determined the clinical significance of serum vascular endothelial growth factor (VEGF) and serum complement 3a (C3a) levels in patients with CRC in Fars province, southern Iran. Materials and Methods: Between June 2010 and June 2012, 110 patients with CRC of both genders and different age groups were divided into 3 groups. Group A included patients who had just undergone surgery; Group B had undergone chemotherapy after surgery; and Group C had undergone chemotherapy and radiotherapy after surgery. Twenty one healthy subjects with normal colonoscopy were considered as a control group. ELISA was undertaken to determine VEGF and C3a levels before and after treatment measures. Results: The mean age of patients was $53.9{\pm}14.1$ years. Considering VEGF level, a significant decrease was visible after treatment measures in groups A and B, but not Group C. For VEGF level, the difference was not statistically significant between two genders and various age groups before and after treatment. No significant difference was found for VEGF level between patients and normal group before any treatment. Regarding C3a levels in 101 subjects, they significantly decreased after treatment measures. Before and after treatment, the difference was statistically significant between two genders, but was not statistically significant among various age groups. Conclusions: As VEGF and C3a levels were significantly lower in patients after treatment, these may be beneficial markers in assessment of CRC therapy especially in early stages.

• 한국동물위생학회지
• /
• 제36권4호
• /
• pp.249-254
• /
• 2013

This study was done to determine the seroprevalence of Coxiella burnetii in cattle with reproductive disorders reared in eastern Gyeongbuk province. A total of 146 cattle blood samples from 83 farms in eastern Gyeongbuk province were collected in 2011~2012. Among them, 17 (11.6%) samples from 11 (13.3%) farms were seropositive for C. burnetii by ELISA. In dairy cattle with mastitis, 15 (38.5%) milk samples from 9 (42.9%) farms were seropositive among 39 milk samples from 21 farms. Among the seropositive farms, one farm with high seroprevalence was selected to assess the seroprevalence of C. burnetii in the bulk-tank milk (BTM) and blood samples of dairy cattle. The BTM sample from the farm was seropositive to C. burnetii and 6 (15%) out of 40 dairy cattle were seropositive. In cattle with reproductive disorders including infertility, abortion and stillbirth, 2 (1.9%) samples from 2 (3.2%) farms were positive among 107 samples collected from 62 farms. Among 2 positive samples, one of them is dariy cattle (100%) and the other is Korean cattle (0.9%) (P<0.001). This suggests that Korean cattle was relatively resistant to C. burnetii. Based on the data obtained, we identified association between C. burnetii shedding in milk and chronic subclinical mastitis in dairy cattle. Since seroprevalence of C. burnetii is relatively high in dairy cattle, further intensive studies on C. burnetii among the high risk milk farms and dairy cattle herds are needed to evaluate infection status.

• Journal of Animal Science and Technology
• /
• 제63권2호
• /
• pp.426-439
• /
• 2021

It is already well known that castration improves marbling quality but exact timing of castration is still highly debated in beef cattle production industry. After castration, blood hormonal changes occur in steer and objective of this study was to investigate the effects of growth hormone (GH) levels on adipocyte differentiation in stromal vascular cells (SVCs) and transdifferentiation into adipocytes in C2C12 myoblasts. Total GH concentrations were measured via enzyme-linked immunosorbent assay (ELISA) in 24 male calves and 4 female calves. Cell proliferation, cellular triglyceride (TG) accumulation, and the cell's lipolytic capability were measured in C2C12 myoblasts and SVCs. Myogenic, adipogenic, and brown adipocyte-specific gene expression was measured via real-time polymerase chain reaction (PCR) using SYBR green. Serum GH levels were the highest in late-castrated calves. Treatment with 5 ng/mL GH resulted in greater TG accumulation as well as increased CCAAT-enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ expression compared to that after treatment with 15 ng/mL GH. Treatment with 5 ng/mL GH also resulted in lower myogenin (myo)G and myoD expression compared to that after treatment with 15 ng/mL GH. The expression of bone morphogenetic protein (BMP) 7 after treatment with 5 ng/mL GH was higher than that after treatment with 15 ng/mL GH. But carcass characteristics data showed no significant difference between early and late castrated steers. Therefore, our results indicate that castration timing does not seem to be inevitable determinate of carcass qualities, particularly carcass weight and marbling score in Hanwoo beef cattle.