• Clinical and Experimental Vaccine Research
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• 제12권4호
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• pp.328-336
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• 2023

Purpose: Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Materials and Methods: Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. Results: With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Conclusion: Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

• BMB Reports
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• 제50권5호
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• pp.263-268
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• 2017

${\beta}$-Agarase cleaves the ${\beta}$-1,4 linkages of agar to produce neoagarooligosaccharides (NAO), which are associated with various physiological functions. However, the immunological functions of NAO are still unclear. In this study, we demonstrated that ${\beta}$-agarase DagA-produced neoagarohexaose (DP6), an NAO product, promoted the maturation of dendritic cells (DCs) by Toll-like receptor 4 (TLR4). DP6 directly and indirectly enhanced the activation of natural killer (NK) cells in a TLR4-dependent manner in vitro and in vivo. Finally, the antitumor activity of DP6 against B16F1 melanoma cells was inhibited in NK cell-depletion systems by using NK-cell depleting antibodies in vivo. Collectively, the results indicated that DP6 augments antitumor immunity against B16F1 melanoma cells via the activation of DC-mediated NK cells in a TLR4-dependent manner. Thus, DP6 is a potential candidate adjuvant that acts as an immune cell modulator for the treatment of melanoma.

• Asian Pacific Journal of Cancer Prevention
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• 제15권3호
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• pp.1321-1326
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• 2014

Background/Aim: Toll-like receptor 4 (TLR4) and B7-H1, both normally expressed restricted to immune cells, are found to be aberrantly expressed in a majority of human tumors and may play important roles in regulation of tumor immunity. It has been shown that urothelial bladder cancer (UBC) patients can manifest tumoral immune escape which may be a potential critical factor in tumor pathogenesis and progression. However, so far, the mechanisms of UBC-related immune escape have not been clarified. The aim of this study was to investigate the effect of TLR4 and B7-H1 on immune escape of UBC. Methods: Bladder cancer T24 cells were pre-incubated with LPS and co-cultured with tumor specific CTLs. CTL cytotoxicity and apoptosis rates were measured by MTT assay and flow cytometry, respectively. The effects of an ERK inhibitor on B7-H1 expression and CTL cytotoxicity against T24 cells were also evaluated. In addition, TLR4, B7-H1 and PD-1 protein expression was analyzed by immunohistochemistry in 60 UBC specimens and 10 normal urothelia. Results: TLR4 activation protected T24 cells from CTL killing via B7-H1 overexpression. However PD98059, an inhibitor of ERK, enhanced CTL killing of T24 cells by reducing B7-H1 expression. TLR4 expression was generally decreased in UBC specimens, while B7-H1 and PD-1 were greatly overexpressed. Moreover, expression of both B7-H1 and PD-1 was significantly associated with UICC stage and WHO grade classification. Conclusions: TLR4 and B7-H1 may contribute to immune escape of UBC. Targeting B7-H1 or the ERK pathway may offer new immunotherapy strategies for bladder cancer.

• Clinical and Experimental Pediatrics
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• 제60권7호
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• pp.208-215
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• 2017

Purpose: Activation of Toll-like receptor 2 (TLR2) present on circulating monocytes in patients with Kawasaki disease (KD) can lead to the production of proinflammatory cytokines and interleukin-10 (IL-10). We aimed to determine the association of the frequency of circulating TLR2+/ CD14+ monocytes (FTLR2%) with the outcomes of KD, as well as to compare FTLR2% to the usefulness of sIL-10. Methods: The FTLR2% in patients with KD was measured by flow cytometry. Serum levels of IL-10 (sIL-10) were determined in 31 patients with KD before the initial treatment with intravenous immunoglobulin (IVIG) and in 21 febrile controls by using enzyme-linked immunosorbent assay. Patients were classified as having coronary artery lesions (CALs) based on the maximal internal diameters of the proximal right coronary artery and proximal left anterior descending coronary artery one month after the initial diagnosis. Results: We found that FTLR2% greater than 92.62% predicted CALs with 80% sensitivity and 68.4% specificity, whereas FTLR2% more than 94.61% predicted IVIG resistance with 66.7% sensitivity and 71.4% specificity. Moreover, sIL-10 more than 15.52 pg/mL predicted CALs and IVIG resistance with 40% and 66.7% sensitivity, respectively, and 73.7% and 76.2% specificity, respectively. Conclusion: We showed that measuring FTLR2% before the initial treatment could be useful in predicting CAL development with better sensitivity than sIL-10 and with results comparable to sIL-10 results for the prediction of IVIG resistance in patients with KD. However, further studies are necessary to validate FTLR2% as a marker of prognosis and severity of KD.

• Journal of Ginseng Research
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• 제46권1호
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• pp.156-166
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• 2022

Background: Panax ginseng Meyer (P. ginseng), a herb distributed in Korea, China and Japan, exerts benefits on diverse inflammatory conditions. However, the underlying mechanism and active ingredients remains largely unclear. Herein, we aimed to explore the active ingredients of P. ginseng against inflammation and elucidate underlying mechanisms. Methods: Inflammation model was constructed by lipopolysaccharide (LPS) in C57BL/6 mice and RAW264.7 macrophages. Molecular docking, molecular dynamics, surface plasmon resonance imaging (SPRi) and immunofluorescence were utilized to predict active component. Results: P. ginseng significantly inhibited LPS-induced lung injury and the expression of proinflammatory factors, including TNF-α, IL-6 and IL-1β. Additionally, P. ginseng blocked fluorescencelabeled LPS (LPS488) binding to the membranes of RAW264.7 macrophages, the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Furthermore, molecular docking demonstrated that ginsenoside Ro (GRo) docked into the LPS binding site of toll like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex. Molecular dynamic simulations showed that the MD2-GRo binding conformation was stable. SPRi demonstrated an excellent interaction between TLR4/ MD2 complex and GRo (K_D value of 1.16 × 10^-9 M). GRo significantly inhibited LPS488 binding to cell membranes. Further studies showed that GRo markedly suppressed LPS-triggered lung injury, the transcription and secretion levels of TNF-α, IL-6 and IL-1β. Moreover, the phosphorylation of NF-κB and MAPKs as well as the p65 subunit nuclear translocation were inhibited by GRo dose-dependently. Conclusion: Our results suggest that GRo exerts anti-inflammation actions by direct inhibition of TLR4 signaling pathway.

• Asian Pacific Journal of Cancer Prevention
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• 제14권4호
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• pp.2295-2299
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• 2013

Background: To explore the role of caveolin-1(CAV-1) gene silencing on MAPK activation in lipopolysaccharide (LPS)-challenged human mammary epithelial cells. Methods: We established a MCF-10ACE of CAV-1 gene silencing from human mammary epithelial cell line MCF-10A by RNAi technology. DNA Microarray were used to detect the expression of inflammation-associated genes in MCF10ACE. Western blotting was used to examine the activation of MAPK in lipopolysaccharide(LPS)-challenged MCF-10A and MCF-10ACE. Moreover, immunofluorescence and Western bloting were performed to detect the co-localization of CAV-1 and toll-like receptor 4 (TLR4) in human mammary epithelial cells. Results: MCF-10ACE exhibited significant increases in inflammation-associated gene expression, especially IL-6 (~7-fold) and IL6R (~17-fold). In addition, LPS-induced p38 MAPK and JNK MAPK activation was significantly increased in MCF-10ACE. Furthermore, CAV-1 co-localized with TLR4 and appeared a negative correlation trend. Conclusion: CAV-1 gene silencing promotes MAPK activation via TLR4 signaling in human mammary epithelial cells response to LPS.

• 한국식품영양학회지
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• 제34권5호
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• pp.441-448
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• 2021

This study was designed to investigate the intracellular signaling pathways and immunoenhancing effect of macrophage activation by crude polysaccharides (CPP) extracted from citrus peels. CPP did not affect the cytotoxicity of RAW264.7 cells, but showed dose-dependent effects on cell viability. Also, CPP showed high production of chemokine (nitric oxide (NO)) and cytokines (interleukin (IL)-6 and tumor necrosis factor (TNF)-α). CPP increased IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) mRNA expression dose-dependently. CPP also strongly induced the phosphorylation of the ERK, p38, and IκBα pathways in RAW 264.7 cells. In anti-pattern recognition receptors (PRRs) experiments, the effect of CPP on NO production was strongly suppressed by neutralizing toll-like receptor (TLR)2, TLR4, and Dectin1 antibodies, whereas IL-6 and TNF-α production by CPP was mainly suppressed by mannose receptor (MR). Therefore, these results suggest that CPP treatment-induced NO production was regulated by the ERK, p38, and NF-κB pathways through TLR2, TLR4, and Dectin1 receptors, whereas IL-6 and TNF-α production was primarily regulated by the ERK, p38, and NF-κB pathways through MR receptors.

• Tuberculosis and Respiratory Diseases
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• 제58권4호
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• pp.367-374
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• 2005

연구배경 : 장기간 흡연을 하는 사람의 10-20%에서만 COPD가 발생한다는 사실은 COPD의 발생에 유전적 인자가 관여함을 시사한다. 최근 surfactant protein A가, COPD의 병인에 중요한 역할을 하는 것으로 알려진 MMP-9의 분비를 TLR2를 통해 증가시킨다고. 그러므로 COPD의 병인에 TLR2이 역할을 할 수 있을 것이라는 가정 아래, TLR2 유전자의 intron II에 존재하는 Guanine-Thymine (GT)의 반복으로 이루어진 유전자다형성과 한국인에서의 COPD의 발생과의 연관성을 규명하고자 하였다. 방 법 : 흡연력이 있는 남자 COPD 환자와 정상 폐기능을 보이는 남자 흡연자를 대상으로 하여, TLR2 유전자의 intron II의 GT 반복횟수를 확인하였다. 그 GT 반복이 3상성의 분포를 보여 이들을 다시 세 개의 맞섬 유전자 아형으로 분류하여 분석하였다. (12-16회 GT 반복: 짧은 아형; 17-22회 반복: 중간 아형; 23-27회 반복: 긴 아형) 결 과 : 각각의 맞섬유전자 아형의 분포는 125명의 COPD군과 144명의 대조군 사이에 유의한 차이는 없었다(P=0.75). 또한 각각의 맞섬유전자 아형의 유무에 따른 유전형의 빈도도 두 군간의 차이는 관찰할 수 없었다. 결 론 : TLR2 유전자의 intron II에 존재하는 GT 반복으로 이루어진 유전자다형성은 한국인에서 COPD의 발생과 연관되어 있지 않다.

• Journal of Ginseng Research
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• 제43권4호
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• pp.606-617
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• 2019

Background: The Panax ginseng berry extract (GBE) is well known to have an antidiabetic effect. The aim of this study is to evaluate and investigate the protective effect of ultrasonication-processed P. ginseng berry extract (UGBE) compared with GBE on liver fibrosis induced by mild bile duct ligation (MBDL) model in rats. After ultrasonication process, the composition ratio of ginsenoside in GBE was changed. The component ratio of ginsenosides Rh1, Rh4, Rg2, Rg3, Rk1, Rk3, and F4 in the extract was elevated. Methods: In this study, the protective effect of the newly developed UGBE was evaluated on hepatotoxicity and neuronal damage in MBDL model. Silymarin (150 mg/kg) was used for positive control. UGBE (100 mg/kg, 250 mg/kg, 500 mg/kg), GBE (250 mg/kg), and silymarin (150 mg/kg) were orally administered for 6 weeks after MBDL surgery. Results: The MBDL surgery induced severe hepatotoxicity that leads to liver inflammation in rats. Also, the serum ammonia level was increased by MBDL surgery. However, the liver dysfunction of MBDL surgery-operated rats was attenuated by UGBE treatment via myeloid differentiation factor 88-dependent Toll-like receptor 4 signaling pathways. Conclusion: UGBE has a protective effect on liver fibrosis induced by MBDL in rats through inhibition of the TLR4 signaling pathway in liver.

• Journal of Microbiology and Biotechnology
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• 제28권6호
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• pp.860-873
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• 2018

Although ginseng marc is a by-product obtained during manufacturing of various commercial ginseng products and has been routinely discarded as a waste, it still contains considerable amounts of potential bioactive compounds, including saponins and polysaccharides. Previously, we reported that ginseng oligosaccharides derived from ginseng marc polysaccharides by enzymatic hydrolysis exert immunostimulatory activities in macrophages and these activated macrophages are in turn able to inhibit the growth of skin melanoma cells by inducing apoptosis. In the present study, a more detailed investigation of the immunostimulatory activity and underlying action mechanisms of an enzymatic hydrolysate (GEH) containing these oligosaccharides derived from ginseng marc polysaccharides was performed. The levels of proinflammatory cytokines and anti-inflammatory cytokines were measured in GEH-stimulated RAW264.7 macrophages using RT-PCR analysis and ELISA. The expression levels of Toll-like receptor 2 (TLR2) and TLR4, Dectin-1, and MerTK were measured by RT-PCR analysis or western blot analysis, and the phagocytic activities of GEH-challenged bone marrow-derived macrophages toward apoptotic Jurkat cells were assayed using fluorescence microscopy. GEH induced the production of both proinflammatory cytokines $TNF-{\alpha}$ and IL-6, and anti-inflammatory cytokine IL-10 in RAW 264.7 cells. The expression of the TLR2 and MerTK mRNAs was increased upon GEH treatment. Phagocytosis of apoptotic Jurkat cells was enhanced in GEH-treated macrophages. Based on the results, this enzymatic hydrolysate (GEH) containing oligosaccharides exerts immunostimulatory effects by maintaining the balance between M1 and M2 cytokines, facilitating macrophage activation and contributing to the efficient phagocytosis of apoptotic cells. Therefore, the GEH could be developed as value-added, health-beneficial food materials with immunostimulatory effects.