• 대한한방소아과학회지
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• 제29권4호
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• pp.39-66
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• 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.

• IMMUNE NETWORK
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• 제13권4호
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• pp.116-122
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• 2013

This study was conducted to determine whether CD4 T cell responses to citrullinated fibrinogen occur in patients with rheumatoid arthritis (RA), especially in HLA-DR4-positive subjects. Whole peripheral blood mononuclear cells (PBMCs) of RA patients and control subjects were stimulated with citrullinated fibrinogen peptides, and T-cell production of proliferation and proinflammatory cytokines, such as interferon-${\gamma}$(IFN-${\gamma}$) and interleukin-17A (IL-17A), were measured. In addition, CD4 T cells from RA patients were stimulated with the citrullinated fibrinogen peptide, $Fib-{\alpha}$ R84Cit, identified as a DRB1*0401-restricted T cell epitope in HLA-DR4 transgenic mice, and the degree of T cell activation was examined similarly. No proliferative responses to the citrullinated fibrinogen peptides were observed in whole PBMCs or CD4 T cells from RA patients. Furthermore, no increased production of IFN-${\gamma}$ or IL-17A was found in whole PBMCs or CD4 T cells stimulated with the citrullinated fibrinogen peptides, although these cells responded to recall antigen, a mixture of tetanus toxoid, purified protein derivative (PPD) from Mycobacterium tuberculosis, and Candida albicans. The results of this study indicate that anti-citrulline immunity in RA patients may be mediated by fibrinogen because there is no evidence of CD4 T cell-mediated immune responses to citrullinated fibrinogen peptides.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2002년도 학술대회지
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• pp.185-211
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• 2002

We have found many beneficial effects of the long-tenn intake of Korean red ginseng (KRG) in human immunodeficiency virus (HIV) type-I infected patients, including the maintenance of CD4+ T cell count for 10 years with KRG only and the delayed development of resistance mutation to ZDV. In this study, to investigate whether KRG-intake could affect the clinical progression and nef gene variation, we determined 200nef sequences from 70 patients. Follow-up period was $8.8{\pm}2.9$ years and annual decrease in CD4+T cell was $41{\pm}57/ul.$ Nested polymerase chain reaction (PCR) and direct sequencing were perfonned with peripheral blood mononuclear cells (PBMC) obtained at times during the study period. First, there was a significant correlation between survival duration and duration of KRG-intake $(36.8{\pm}38$ months)(P=0.000). There were significant correlations between the last NefProg score and CD4+ T cell count (r= 0.208, P<0.05) and annual decrease in CD4+ T cell count (r =0.346, P<0.01) in 70 patients. In addition, there were significant correlations between KRG-intake and annual decrease (r= 0.323, P<0.01), and the CD4+ T cell count itself (r=0.229, p<0.05). Furthennore, there was also a mild significance between the NefProg score and the duration of KRG-intake in only SP and RP (n=30, r=-0.281, P=0.067). In addition, we detected various defects in 21 patients $(30.0\%),$ not including 5 premature stop codons. Ten $(12.5\%)$ patients showed repeated deletion of an amino acid. Four of 10 patients were gross deletions and they were treated with KRG for more than 20 months. The number of patients with repeated gross deletions was significantly higher in the order of slow progressors $(18\%)$, typical progressors($3\%$), and rapid progressors($0\%$) (P<0.05). We also observed that long-tenn intake of KRG might make the change from A or D to T at position 54 and decrease NefProg score. Taken together, our results show clear evidence that the long-term intake of KRG has effects on nef gene variation as well as definite clinical usefulness.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제24권4호
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• pp.377-383
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• 2021

Purpose: We investigated the association of effector memory (EM) CD8⁺ T cell and CD4⁺ T cell immunity with metabolic syndrome (MS). Methods: Surface and intracellular staining of peripheral blood mononuclear cells was performed. Anti-interleukin-7 receptor-alpha (IL-7Rα) and CX3CR1 antibodies were used to stain the subsets of EM CD8⁺ T cells, while anti-interferon-gamma (IFN-γ), interleukin-17 (IL-17), and forkhead box P3 (FOXP3) antibodies were used for CD4⁺ T cell subsets. Results: Of the 47 obese children, 11 were female. Children with MS had significantly higher levels of serum insulin (34.8±13.8 vs. 16.4±6.3 µU/mL, p<0.001) and homeostasis model assessment of insulin resistance (8.9±4.1 vs. 3.9±1.5, p<0.001) than children without MS. Children with MS revealed significantly higher frequencies of IL-7Rα^low CD8⁺ T cells (60.1±19.1% vs. 48.4±11.5%, p=0.047) and IL-7Rα^lowCX3CR1⁺ CD8⁺ T cells (53.8±20.1% vs. 41.5±11.9%, p=0.036) than children without MS. As the serum triglyceride levels increased, the frequency of IL-7Rα^lowCX3CR1⁺ and IL-7Rα^highCX3CR1^- CD8⁺ T cells increased and decreased, respectively (r=0.335, p=0.014 and r=-0.350, p=0.010, respectively), in 47 children. However, no CD4⁺ T cell subset parameters were significantly different between children with and without MS. Conclusion: In obese children with MS, the changes in immunity due to changes in EM CD8⁺ T cells might be related to the morbidity of obesity.

• 대한의생명과학회지
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• 제20권4호
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• pp.250-255
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• 2014

Mycobacterium tuberculosis (MTB) resides and replicates inside macrophages. In our previous report, we reported that CD8+ T cell-mediated immune responses specific for the peptide derived from MTB RNA polymerase beta-subunit ($RpoB_{127-135}$) could be induced in TB patients expressing HLA-$A^*0201$ subtype. In order to examine whether $RpoB_{127-135}$ specific CD8+ T cells can recognize MTB infected macrophages in vitro, CD8+ T cell lines specific for $RpoB_{127-135}$ peptide were generated from peripheral blood mononuclear cells (PBMCs) of healthy HLA-$A^*0201$ subjects by in vitro immunization technique. In this study, we observed $RpoB_{127-135}$ specific CD8+ T cells could recognize and destroy macrophages infected with MTB for 2 to 4 days. $RpoB_{127-135}$ specific CD8+ T cell immune response was inducible from PBMC of healthy subjects expressing HLA-$A^*0206$ subtype, one of HLA-A2 supertype members. Next, we investigated the HLA-I processing mechanism of $RpoB_{127-135}$ peptide in MTB infected macrophages. As a result, the presentation of the MTB derived epitope peptide, $RpoB_{127-135}$, to CD8+ T cells was not inhibited by the treatment with brefeldin-A (ER-Golgi transport inhibitor) or lactacystin (proteasome inhibitor), which blocks the classical HLA-I processing pathway. However, $RpoB_{127-135}$ specific CD8+ T cell activity was blocked either by the blocking agent for the endocytosis (cytochalasin D) or by the blocking antibody (W6/32) for HLA-I molecules. Therefore, the $RpoB_{127-135}$ peptide may be processed by accessing the alternate HLA-I processing pathway. Understanding the processing and presentation mechanisms of the MTB derived proteins will help to improve the efficacy of vaccines and the efficiency of therapeutic agents for TB.

• 대한한의학회지
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• 제27권4호
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• pp.1-11
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• 2006

Objectives : We investigated the effect of Dohongsamul-tang (DHSMT) on the production of various cytokines in lipopolysaccaride (LPS) stimulated peripheral mononuclear cells (PBMCs) from CI patients. Methods: Cell viability was determined using MTT assay. ELISA was carried out for investigating $TNF-{\alpha}$, $IL-1{\beta}$, IL-6, IL-8, IL-4, and $TGF-{\beta}$ 1 Results : The amount of tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-$1{\beta}$, IL-6, IL-8, IL-4, and transforming growth factor(TGF)-${\beta}$ 1 in PBMC culture supernatant significantly increased in the LPS treated cells compared to unstimulated cells. We show that DHSMT inhibited the production of TNF-${\alpha}$, IL-1${\beta}$, IL-6, IL-8, and IL-4 induced by LPS in a dose dependent manner. The maximal inhibition rate of the TNF-${\alpha}$, IL-1${\beta}$, IL-6, IL-8, and IL-4 production by pretreatment of DHSMT (1.0mg/ml) was 38.52 ${\pm}$ 2.5% (P < 0.01), 44.02 ${\pm}$ 3.5% (P < 0.05), 45.32 ${\pm}$ 2.3% (P < 0.01), 42.30 ${\pm}$ 3.1% (P < 0.05), and 49.70　${\pm}$　3.1%(P ＆lt; 0.05), respectively. On the other hand, DMSMT significantly increased the LPS-induced TGF-${\beta}$ 1 production(P<0.05). Conclusions : Taken together. these results suggest that DHSMT might have regulatory effects on cytokine production, which might explain its beneficial effect in the treatment of CI.

• Tuberculosis and Respiratory Diseases
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• 제48권2호
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• pp.223-235
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• 2000

배 경: 액체환기가 손상된 폐에 긍정적인 영향을 주는 한 기전으로 perfluorocarbon(PFC)이 기도상피세포에서 chemokine발현을 억제할 수 있는 지를 관찰하기 위해 본 연구를 시행하였다. 방 법: 기도 상피세포로는 A549 세포주를, PFC로는 perfluorodecalin을, PFC의 노출은 Transwell$^{(R)}$배양접시의 lower chamber를 이용하여 시행하였다. PFC가 말초혈액 탄핵구층(peripheral blood mononuclear cell : PBMC)의 기능을 억제해서 A549세포의 chemokine 발현을 억제할 수 있는지를 관찰하기 위해서 PBMC를 분리하여 Transwell$^{(R)}$ 접시에서 배양하면서 lipopolysaccharide(LPS, 10 ${\mu}g/mL$)로 자극과 PFC의 노출에 따라 군을 나누었으며 24시간 후 그 배양 상층액을 포함한 conditioned media(CM)으로 24시간 동안 A549세포를 자극한 후 chemokine발현을 측정하였다. 또한 PFC가 직접 기도 상피세포의 기능을 억제할 수 있는 지를 관찰하기 위해서 A549 세포를 Transwell$^{(R)}$ 접시에서 배양하면서 interleukin-l$\beta$(IL-1$\beta$, 10 ng/ml), TNF-$\alpha$(10 ng/ml)로 각각 혹은 동시에 24시간동안 자극하면서 PFC노출여부에 따른 IL-8과 RANTES발현 정도를 비교하였다. Chemokine 발현은 IL-8과 RANTES의 단백에 대한 ELISA와 mRNA는 Northern analysis를 통하여 분석하였다. 결 과: 1. LPS로 자극한 PBMC의 배양상층액을 포함한 CM로 A549세포를 자극하였을 때 IL-8과 RNATES mRNA 발현과 면역반응성 단백 생성이 의미 있게 상승하였으나(p<0.05) PFC노출여부에 따른 유의한 차이는 관찰할 수 없었다. 2. TNF-$\alpha$와 IL-1$\beta$ 모두 A549세포에서 IL-8과 RANTES mRNA자발현과 면역반응성 단백 생성의 증가를 가져왔으나(p<0.05) PFC노출에 따른 유의한 차이는 관찰할 수 없었다. 결 론: 기도 상피세포의 chemokine발현 감소를 통한 항염증 작용은 액체환기시 보이는 염증반응 감소에 큰 기여를 하지 않을 것으로 생각되며 추후 액체환기시 관찰되는 염증반응의 감소의 기전에 대한 연구가 더 필요할 것으로 사료된다.

• Tuberculosis and Respiratory Diseases
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• 제80권1호
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• pp.77-82
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• 2017

Background: Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens. Methods: Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured. Results: CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-${\gamma}$ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor ${\alpha}$ and interleukin 10, respectively. Conclusion: ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis.

• 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.

• 대한한방소아과학회지
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• 제25권1호
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• pp.1-27
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• 2011

Objectives: The purpose of this study is to investigate the effects of Yijungtang(YJT) on atopic dermatitis in an in-vitro and in-vivo experiment using a RBL-2H3 mast cells and a NC/Nga atopic dermatitis mouse. Methods: In-vitro experiment, IL-4, IL-13 mRNA expression were evaluated by a real-time PCR, IL-4, IL-13 production by ELISA and transcription factor as GATA-1, GATA-2, NF-AT1, NF-AT2, AP-1 and NF-kB by western blotting. In-vivo experiment, clinical skin score we evaluated by, hematology and Serum total IgE and IgG1 of NC/Nga atopic dermatitis mouse, cytokine level, total number of cell, Immunohistochemical staining and Histological features of auxiliary lymph node(ALN), draining lymph node(DLN), peripheral blood mononuclear cells(PBMCs) and dorsal skin tissue in NC/Nga mouse. Results: YJT decreased IL-4, IL-13 mRNA expression, IL-4, IL-13 production and prominently decreased the expression of mast cell specific transcription factors including GATA-2, NF-AT2, c-Fos and NF-kB. YJT oral administration reduced the levels of skin severity scores. It also decreased the level of inflammatory cytokines such as IL-5, IL-13, histamine and IgE in the serum. It elevated IFN-gamma level in the spleenocyte culture supernatant but decreased. $CD3e^+$, $CD19^+$, $CD4^+$, $CD8^+$, $CD3e^+CD69^+$, $CD11b^+Gr-1^+$, $CCR3^+$ in the PBMCs, $CD4^+$, $CD8^+$, $CD3e^+CD69^+$, $B220^+CD23^+$ in the ALN, $CD4^+$, $CD3e^+CD69^+$ in the ALN and $CD4^+$, $CD11b^+Gr-1^+$ in the dorsal skin. Histological examination showed that infiltration levels of immune cells in the skin of AD-induced NC/Nga mice were much improved by YJT oral administration. Conclusions: The anti-allergic activities of YJT may be mediated by down-regulation of Th2 cytokines, such as IL-4 and IL-13, through the regulation GATA-2, NF-AT2 and NF-kB transcription factors in mast cells. YJT would be regulate molecular mediators and immune cells which are functionally associated with atopic dermatitis induced in NC/Nga mice, and may play an important role in recovering AD symptoms.