• 약학회지
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• 제54권3호
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• pp.192-199
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• 2010

Colorectal cancer is one of the most common alimentary malignancies. In this study, the antitumor activity of activated human natural killer (NK) cells against human colorectal cancer was evaluated in vivo. Human NK cells are the key contributors of innate immune response and the effective functions of these cells are enhanced by cytokines. Human peripheral blood mononuclear cells (PBMC) were cultured with interleukin-2 (IL-2)-containing medium for 14 days and resulted in enriched NK cell population. The resulting populations of the cells comprised 7% $CD3^+CD4^+$ cells, 25% $CD3^+CD8^+$ cells, 13% $CD3^-CD8^+$ cells, 4% $CD3^+$CD16/$CD56^+$ cells, 39% $CD3^+$CD16/$CD56^-$ cells, and 52% $CD3^-$CD16/$CD56^+$ cells. Tumor necrosis factor alpha (TNF-$\alpha$), interferon gamma (IFN-$\gamma$), IL-2, IL-4, and IL-5 transcripts of the activated NK cells were confirmed by RT-PCR. In addition, activated NK cells at doses of 2.5, 5 and 10 million cells per mouse inhibited 10%, 34% and 47% of SW620-induced tumor growth in nude mouse xenograft assays, respectively. This study suggests that NK cell-based immunotherapy may be used as an adoptive immunotherapy for colorectal cancer patients.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.113-113
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• 1995

본 연구는 표고버섯에서 분리정제한 렉틴 성분(LEL)을 말초혈액 단핵세포(PBMC)에 반응시켜 사이토카인 유도능을 지질다당류(LPS)와 비교하여 역전사효소 중합반응법(RT-PCR)으로 측정하였다. 측정 대상 사이토카인은 IL-1, IL-2, IL-6, TNF$\alpha$ 및 IFN${\gamma}$의 다섯가지였으며 이들을 대상으로 PBMC에 렉틴을 적용하여 1, 8, 24, 48, 72, 96, 120분 등의 시간대에서 반응시켜 사이토카인의 유전자 발현 유도에 관한 다음의 결과를 얻었다. LEL의 사용 농도에 따른 편도선 림프구(tonsillar lymphocyte)의 TNF$\alpha$ 유전자 발현 양상은 반응 1시간의 경우 LEL의 일부 농도와 LPS 전농도에서 관찰되었으나 반응 40시간째에는 LEL 전농도와 LPS 전농도에서 TNF$\alpha$ 유전자 발현 양상을 관찰할 수 없었다. RT-PCR 결과 원액이나 회석액 재료로부터 관찰된 TNF$\alpha$유전자 band의 강 약 차이는 나타나지 않았다. LEL의 자극에 의한 반응 시간대 별 PBMC에 의한 사이토카인 유전자 발현 양상은 위에서 언급된 다섯가지 사이토카인을 유도, 생성할 수 있다는 것이 확인되었는데, IL-2, IL-6 및 IFN${\gamma}$는 120시간까지 장시간 지속되는 유전자 발현이 가능한 반면 TNF $\alpha$의 생성 양상은 이들 사이토카인의 생성 양상과는 판이하게 반응 1, 8 및 24 시간대까지만 TNF$\alpha$ 유전자 발현을 관찰할 수 있었고 IL-1은 72 시간까지 반응을 나타내는 등 특이적 양상을 보였다. 한편 LPS는 실험에 사용된 전 사이토카인의 유전자 발현을 120시간대까지 반응이 유지됨을 관찰하였기에 LPS가 PBMC의 강력한 사이토카인 유도체임을 입증할 수 있었으며 LEL과 다소 상이한 결과를 보였으나 LEL 또한 PBMC로부터 사이토카인을 생성 유지시킬 수 있는 유도체로 작용함을 확인할 수 있었다.

• 대한한방내과학회지
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• 제21권3호
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• pp.433-441
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• 2000

Objective : To analyze the effects of Yukmijihwang-tang, Taeksa-tang, Silbi-um on mesangial cell proliferation, fibronectin synthesis and MHC-class II expression. Methods : Laboratory studies were performed with the method of surface enzyme immunoassays or flow cytometry after addition of peripheral blood mononuclear cells(PBMC) supernatants treated with medications using the cultured human mesangial cells. Results : 1. Silbi-um produces more suppressive effect than control group and hydrocortisone group on the mesangial cell proliferation. In Yukmijihwang-tang, Taeksa-tang and Silbi-um, mesangial cell proliferation significantly decreased than in hydrocortisone group 2. In the 'without fetal bovine serum' study, Yukmijihwang-tang take more suppressive effect than Control group on the fibronectin synthesis. In the 'with fetal bovine serum' study, Yukmijihwang-tang, Taeksa-tang, Silbi-um all have suppressive effect, but it hasn' t any statistical significance. 3. Yukmijihwang-tang, Taeksa-tang, Silbi-um all have a suppressive effect on the MHC-class II expression. Conclusions : Herb medicine generally show a suppressive effect on the suppression of the mesangial cell proliferation, fibronectin synthesis and MHC-class II expression.

• 대한바이러스학회지
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• 제29권2호
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• pp.107-118
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• 1999

To characterize the molecular nature of human immunodeficiency virus (HIV)-1, we determined the full-length HIV-1 sequences from cultured peripheral blood mononuclear cells (PBMC) of a Korean long-term nonprogressor (LTNP). Without antiretroviral therapy, the individual has maintained CD4+ T counts over $500/{\mu}l$ from 1989 to 1999. Plasma viral RNA copy was 992 U/ml in 1998. Culture supernatant showed positive from culture days 9. A series of 9 overlapping PCR products were amplified from cultured PBMC and cloned About 9.2 kb from R of 5' LTR to R of 3' LTR was determined by automated sequencing. The G-to-A hypermutations were shown throughout the entire region. As a result of G to A hypermutations, premature stop codon was found in integrase coding region. Though there was no recombination between subtypes over all genomes, TATA box in both LTRs was TAAAA which is detected in subtype E instead of TATAA in subtype B. And, there were nucleotide GC insertion between $NF-{\kappa}B$ I and Sp1 III, and duplication of $TCF-1{\alpha}$ in LTR. We could not find any deletion of amino acid in Nef, Gag, Pol and Env gene. This study is the first report on molecular nature of full genomes of HIV-1 isolated in Korea.

• Advances in Traditional Medicine
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• 제2권1호
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• pp.41-46
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• 2002

In our study, the several cytokines were determined in phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) of Adamantiades-Behcets patients. Adamantiades-Behcets disease (ABD) is a systemic inflammatory disorder and might involve immune dysfunction. High levels of $TNF-\alpha,\;IL-1\beta$ and $IFN-{\gamma}$ indicate the activation of inflammatory reactions and immune system in ABD. Gami-Phedoc-San (GPS) is an Oriental herbal medication, which has been used in Korea for the treatment of ABD. GPS (1 mg/ ml) significantly inhibited the secretion of proinflammatory cytokines, $TNF-\alpha\;and\;IL-1\beta$, compared to absence of GPS (by $50.5{\pm}1.9%$ inhibition for $TNF-\alpha$ and $106.9{\pm}16.8%$ for $IL-1\beta$). GPS also inhibited the production of $IFN-\gamma$, immunoregulatory Th1 cytokine, by $78.4{\pm}2.8%$. The inhibitory effects of GPS on cytokine secretion showed dose-dependent manner, and the pre-treatment of 1 mg/ml GPS had better effects than immunosuppressive drug for treatment of ABD, cyclosporin A. Our results suggest that GPS treatment for ABD patients might have pharmacological activity of immune and inflammatory responses through the cytokine modulation.

• Journal of Periodontal and Implant Science
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• 제44권5호
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• pp.235-241
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• 2014

Purpose: Regulatory T cells (Tregs), expressing CD4 and CD25 as well as Foxp3, are known to play a pivotal role in immunoregulatory function in autoimmune diseases, cancers, and graft rejection. Dendritic cells (DCs) are considered the major antigen-presenting cells (APCs) for initiating these T-cell immune responses, of which $CD103^+$ DCs are derived from precursor human peripheral blood mononuclear cells (PBMCs). The aim of the present study was to evaluate the capacity of these PBMC-derived $CD103^+$ DCs to promote the differentiation of antigen-specific Tregs. Methods: Monocyte-derived DCs were induced from $CD14^+$ monocytes from the PBMCs of 10 healthy subjects. Once the $CD103^+$ DCs were purified, the cell population was enriched by adding retinoic acid (RA). Peptide numbers 14 and 19 of Porphyromonas gingivalis heat shock protein 60 (HSP60) were synthesized to pulse $CD103^+$ DCs as a tool for presenting the peptide antigens to stimulate $CD3^+$ T cells that were isolated from human PBMC. Exogenous interleukin 2 was added as a coculture supplement. The antigen-specific T-cell lines established were phenotypically identified for their expression of CD4, CD25, or Foxp3. Results: When PBMCs were used as APCs, they demonstrated only a marginal capacity to stimulate peptide-specific Tregs, whereas $CD103^+$ DCs showed a potent antigen presenting capability to promote the peptide-specific Tregs, especially for peptide 14. RA enhanced the conversion of $CD103^+$ DCs, which paralleled the antigen-specific Treg-stimulating effect, though the differences failed to reach statistical significance. Conclusions: We demonstrated that $CD103^+$ DCs can promote antigen-specific Tregs from naive T cells, when used as APCs for an epitope peptide from P. gingivalis HSP60. RA was an effective reagent that induces mature DCs with the typical phenotypic expression of CD103 that demonstrated the functional capability to promote antigen-specific Tregs.

• Tuberculosis and Respiratory Diseases
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• 제42권5호
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• pp.703-712
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• 1995

연구배경: 말초혈액 단핵구는 정상적인 면역반응에 중요한 세포로서 탐식 기능과 항원 제공 기능외에 다양한 종류의 Cytokine을 생성하며, 이중 IL-8은 호중구에 대한 강력한 주화 작용과 직접적인 활성화 작용 등을 통해서 염증 작용을 증폭시키는 역할을 하는 것으로 알려져 었다. 따라서 IL-8 작용의 억제를 통하여 항염 효과를 기대할 수 있으며, 이러한 작용을 나타낼 것으로 기대되는 Dexamethasone, $PGE_2$, Indomethacin 및 IFN-$\gamma$를 이용하여 이러한 약제들이 말초혈액 단핵구에서 내독소 자극에 의한 IL-8의 생성 자극 효과에 어떤 영향을 미치는 지에 대해서 알아보았다. 방법: 말초혈액 단핵구는 자원자에게서 채취한 혈액을 Ficoll Hypaque density gradient method에 의해서 백혈구 층을 분리한 뒤에 플라스틱 접시에 부착함으로써 분리하였다. Dexamethasone, $PGE_2$, Indomethacin은 내독소로 자극하기 한 시간 전, 후에 처치하였고, IFN-$\gamma$는 내독소로 자극하기 한 시간 전에만 전처치 하였다. 내독소로 자극한 뒤 4시간 뒤에 RNA를 extraction한 뒤 Northern blot analysis를 시행하여 IL-8 mRNA를 분석하였고, 24시간 뒤에 ELISA를 통하여 IL-8 단백을 정량하였다. 이 과정을 3회 반복하여 다음과 같은 결과를 얻었다. 결과: 1) Dexamethasone의 전, 후 처치는 내독소 자극에 의한 IL-8 mRNA와 단백의 생성을 감소시켰다. 2) IFN-$\gamma$의 단독 투여는 내독소 자극을 받지 않은 군에서 IL-8 mRNA와 단백의 생성을 감소시켰다. 3) 내독소 투여 전에 IFN-$\gamma$를 전 처치 하였을 때, 내독소 단독 투여군에 비해 IL-8 mRNA 발현은 감소하였으나 IL-8 단백의 분비에는 차이가 없었다. 4) $PGE_2$와 Indomethacin은 각각 사용한 농도($10^{-6}M$, $10{\mu}M$)에서는 내독소 자극에 의한 IL-8 mRNA 및 단백의 생성에 별다른 영향을 미치지 못하였다. 결론: Dexamethasone은 말초혈액 단핵구에서 내독소의 자극에 의한 IL-8 mRNA 발현 및 단백의 생성을 억제함으로써 염증 반응을 억제할 것으로 기대된다.

• 대한한의학회지
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• 제21권3호
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• pp.40-50
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• 2000

Objectives : The progression of renal disease can be identified as a glomerulosclerosis by histological examination, and the basic mechanism of glomerulosclerosis is mesangial cell proliferation and mesangial matrix accumulation. ICAM-1, ${\beta}1-integrin$ and MHC-class II are known to attribute to the progression of glomerulosclerosis. They mediate cell-cell or cell-matrix interactions and are expressed in response to injury and inflammation. Up to now, there have been few satisfactory regimens to treat glomerular diseases except minimal change nephrotic syndrome, which can be improved by steroid therapy. Studies were performed in order to investigate whether Jinmu-tang has suppressive effects on some factors associated with the progression of glomerular disease, mesangial cell proliferation, fibronectin synthesis, ICAM-1, ${\beta}1-integrin$ and MHC-class II expression. Methods : Studies were performed with the method of surface enzyme immunoassays or flow cytometry after addition of peripheral blood mononuclear cells(PBMC) supernatants treated with Jinmu-tang, using the cultured human mesangial cells. Results : 1. The suppressive effect of Jinmu-tang on mesangial cell proliferation was higher than that of hydrocortisone. 2. Jinmu-tang has some suppressive effects on fibronectin synthesis, ICAM-1, expression, ${\beta}1-integrin$ expression and MHC-class II expression of mesangial cells, but was lower than hydrocortisone. Conclusions : Jinmu-tang generally shows some immunosuppressive effects. We carefully suggest that the above prescription may be applied to prevent the progression of renal disease or can be used as an adjuvant of or a substitute for steroid therapy.

• 생약학회지
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• 제32권1호통권124호
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• pp.31-38
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• 2001

A lectin was purified from Allomyrina dichotoma (ADL) by physiological saline extraction, ammonium sulfate fractionation, anion exchange column chromatography on DEAE Sephadex A-50 and gel filtration column chromatography on Sephadex G-200. Several biochemical properties of ADL were characterized as follows: ADL from gel filtration column chromatography showed single band on SDS-PAGE. ADL agglutinated the erythrocytes of rabbit and human A, B, O, AB. Agglutinability was relatively stable at basic pH, and was stable at temperature below $40^{\circ}C$. Agglutinability was not affected by metal ions and EDTA. This lectin was proved to be a glycoprotein which contains 0.47% of sugars. The molecular weight of ADL was estimated to be 97,000 dalton by SDS-PAGE. By amino acid analysis, ADL exhibited high amounts of aspartic acid. The lectin's immunomodulating effect was measured as cytokine production. The productions of 5 cytokines $(IL-1{\alpha},\;IL-2,\;IL-6,\;IFN{\gamma}\;and\;TNF{\alpha})$ from peripheral blood mononuclear cells were measured by ELISA. The lectin induced the highest secretion of IL-2 at 8 hr, $TNF{\alpha}$ at 4 hr, and $IFN{\gamma}$ at 24hr, respectively. These results suggest that ADL can elicit the production of detectable cytokines from PBMC.

• 생약학회지
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• 제36권2호통권141호
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• pp.129-135
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• 2005

Lectins from Allomyrina dichotoma (ADL) and Bombyx mori (BML) were partially purified by physiological saline extraction, ammonium sulfate fractionation, anion exchange column chromatography on DEAE Sephadex A-50 and gel filtration column chromatography on Sephadex G-200. An assay for cytokine expression was carried out by using reverse transcription polymerase chain reaction(RT-PCR). mRNA isolated from PBMC(human peripheral blood mononuclear cells) were stimulated with ADL(O.D.=0.2) and BML(O.D.=0.1) for various times(1,4,8,24,48 and 72 h) and various cytokine mRNA assessed by RT-PCR were shown as follows: The patterns of bands for IL-1 mRNA of BML were very similar with those from ADL and these bands were decreased along the increasing reaction times after showing a strong band at 1 h. However mRNA expressions for IL-2, IL-6, $IFN{\gamma}$ and $TNF{\alpha}$ showed different patterns between ADL and BML. With the effect of ADL, the expression of IL-2 and IL-6 mRNA were continuously detected until 72 h with the strongest band of IL-2 mRNA at 24 h. The strong bands of $IFN{\gamma}$ mRNA were observed from 4 to 8 h but the strongest one of $TNF{\alpha}$ was just observed at 1 h. Meanwhile with BML, the bands for IL-2 and $IFN{\gamma}$ were increased along the increasing reaction times until 72 h. The strongest bands were showed from 4 to 8 h with IL-6 and at 8 h with $TNF[\alpha}$. To verify quantitatively ELISA was used for assay of protein secretions of the cytokine gene with IL-2 and $IFN{\gamma}$ expressed markedly different in RT-PCR. The highest cytokine secretion for IL-2 was demonstrated at 48 h. The production of $IFN{\gamma}$ was markedly increased at 24 h and secreted highest at 72 h. These result suggest that ADL and BML, as inducers of cytokines, can elicit detectable cytokine mRNA from PBMC within the first few hours of stimulation and maintain the production of cytokines for a few days by the methods of RT-PCR and ELISA.