Fucoidan Suppresses Prostaglandin E2 Production and Akt Activation in Lipopolysaccharide-Stimulated Porcine Peripheral Blood Mononuclear Cells

박근태,안창환,강병택,강지훈,정의배,양만표 한국임상수의학회 2017 한국임상수의학회지 Vol.34 No.3

Fucoidan, a cell wall polysaccharide found in the brown seaweed, is reported to have broad-spectrum biological activities. The objectives of this study were to examine the effect of fucoidan on prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated porcine peripheral blood mononuclear cells (PBMCs) and to determine whether these effects are involved in Akt activation. The levels of PGE2 production in the culture supernatants from PBMCs were determined by the enzyme-linked immunosorbent assay (ELISA) kit and the levels of COX-2 mRNA were measured by real time polymerase chain reaction (RT-PCR). Akt activity was determined by Western blot analysis. Fucoidan in LPS-naïve PBMCs has no effect on PGE2 production and COX-2 mRNA expression. Furthermore, fucoidan does not affect Akt activation in LPS- naïve PBMCs. However, PGE2 production and COX-2 mRNA expression on PBMCs were remarkably enhanced by LPS stimulation. Akt activity was also increased by LPS. Increasing effects of PGE2 production and COX-2 mRNA expression in PBMCs induced by LPS were suppressed by addition of fucoidan. In addition, fucoidan reduced an increase in Akt activity in LPSstimulated PBMCs. These results suggested that fucoidan exerts potent anti-inflammatory properties by suppression of PGE2 production, COX-2 mRNA expression and Akt activation in LPS-stimulated PBMCs.

Effects of Fucoidan on Nitric Oxide Production and Activator Protein-1 Activation in Lipopolysaccharide-Stimulated Porcine Peripheral Blood Mononuclear Cells

박종찬,안창환,강병택,강지훈,정의배,양만표 한국임상수의학회 2015 한국임상수의학회지 Vol.32 No.4

Fucoidan which is sulfated polysaccharide extracted from brown seaweed has a wide variety of internal biological activities. The objectives of this study were to examine the effect of fucoidan on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated porcine peripheral blood mononuclear cells (PBMCs) and to investigate whether this effect is involved in the expression of inducible nitric oxide synthase (iNOS) and the activation of activator portein- 1 (AP-1). The levels of NO production and AP-1 activity in the culture supernatants from porcine PBMCs were measured by the enzyme-linked immunosorbent assay and the levels of iNOS and AP-1 mRNA were determined by real time polymerase chain reaction. Fucoidan in LPS-naïve PBMCs has no effects on the production of NO and activity of AP-1. Expressions of iNOS and AP-1 mRNA in LPS-naïve PBMCs were also not affected by treatment of fucoidan. However, NO production, AP-1 activity and expressions of iNOS and AP-1 mRNA were dramatically increased in PBMCs stimulated with LPS. Enhancing effects of NO production and AP-1 activity in PBMCs induced by LPS were reduced by addition of fucoidan. Fucoidan also inhibited an increase in expressions of iNOS and AP-1 mRNA in LPS-stimulated PBMCs. These results suggested that fucoidan exerts anti-inflammatory effect by down-regulating production of NO via suppressing expression of iNOS and activity of AP-1 in LPS-stimulated porcine PBMCs.

Zinc Enhances Neutrophil Extracellular Trap Formation of Porcine Peripheral Blood Polymorphonuclear Cells through Tumor Necrosis Factor-Alpha from Peripheral Blood Mononuclear Cells

허주행,김학현,강병택,양만표 한국임상수의학회 2020 한국임상수의학회지 Vol.37 No.5

Neutrophil extracellular trap (NET) formation is an immune response for the invasion of microbes. The purpose of this study is to examine the effect of zinc on NET formation of porcine peripheral blood polymorphonuclear cells (PMNs). The NET formation of PMNs was measured by fluorescence microplate reader. The production of tumor necrosis factor (TNF)-α in the culture supernatants from zinc-treated peripheral blood mononuclear cells (PBMCs) was measured by enzyme-linked immunosorbent assay (ELISA). Zinc itself did not have no effect on NET formation. However, the NET formation of PMNs was increased by culture supernatants from PBMCs treated with zinc. Also, the NET formation of PMNs was increased by recombinant porcine (rp) TNF-α. The production of TNF-α in PBMCs culture supernatants was shown to increase upon zinc treatments. These NET formations of PMNs increased by either culture supernatant from PBMCs treated with zinc or rpTNF-α were inhibited by treatment of anti-rpTNF-α polyclonal antibody (pAb). These results suggested that zinc has an immunostimulating effect on the NET formation of PMNs, which is mediated by TNF-α released from zinc-treated PBMCs. Therefore, zinc may play an important role for NET formation in the defense of porcine inflammatory diseases.

The In Vitro Genotoxic Effect of Tucuma (Astrocaryum aculeatum), an Amazonian Fruit Rich in Carotenoids

Olmiro Cezimbra de Souza Filho,Michele Rorato Sagrillo,Luiz Filipe Machado Garcia,Alencar Kolinski Machado,Francine Cadona,Euler Esteves Ribeiro,Marta Maria Medeiros Frescura Duarte,Ademir Farias More 한국식품영양과학회 2013 Journal of medicinal food Vol.16 No.11

Tucuma (Astrocaryum aculeatum) is an Amazonian fruit that presents high levels of carotenoids and other bioactive compounds such as quercetin. The extracts of tucuma peel and pulp present strong antioxidant activity which illustrate an elevated concentration that causes cytotoxic effects in human peripheral blood mononuclear cells (PBMCs). This study performed additional investigations to analyze the potential genotoxic effects of the tucuma extracts on PBMCs. The genotoxicity was evaluated by DNA fragmentation, Comet assay, and chromosomal instability G-band assays. The acute tucuma extract treatment showed genoprotective effects against DNA denaturation when compared with untreated PBMC cells. However, in the experiments with 24 and 72 h treatments to tucuma treatments, we observed low genotoxicity through a concentration of 100 lg/mL, some genotoxic effects related to intermediary concentrations (100–500 lg/mL), and more pronounced genotoxic effects on higher tucuma extract concentrations. After 24 h of treatment, the reactive oxygen species were similar among treatments and PBMC control groups. However, the caspase-1 activity related to the apoptosis and pyroptosis process increased significantly in higher tucuma concentrations. In summary, tucuma extracts, despite their higher antioxidant content and antioxidant activity, would present PBMCs genotoxic effects that are dependent on concentration and time exposition. These results need to be considered in future in vitro and in vivo studies of tucuma effects.

Inhibitory effects of sesamin on human osteoclastogenesis

Orawan Wanachewin,Peraphan Pothacharoen,Prachya Kongtawelert,Thanyaluck Phitak 대한약학회 2017 Archives of Pharmacal Research Vol.40 No.10

The promotional nature of sesamin on humanosteoblast differentiation has been proven. Here, the effectof sesamin on human osteoclasts was investigated in termsof differentiation and function in M-CSF and RANKLinduced human PBMCs. Sesamin treatment significantlydecreased the number of differentiated osteoclastsobserved by TRAP staining; however, sesamin inhibitiondid not result from the alteration of precursor cell proliferation. Sesamin did not decrease NFATc1 gene expression,which opposed the decreasing trend of CathK andTRAP expression. DC-STAMP, but not Atp6v0d2, alsosignificantly decreased in the presence of 14 lM sesamin. Expressions of CCR2b and CCR4 as chemokine receptorswere significantly down-regulated. Sesamin might mediatethe inhibition of human osteoclast differentiation, therecruitment of precursor cells and F-actin formation. Decrease in the area of the resorption pits and the collagenreleased from the bone slices under sesamin treatmentemphasized the inhibitory effects on both the differentiationand function of osteoclasts. Sesamin is a promisingphytochemical agent inhibiting osteoclast differentiationand function.

Single-cell transcriptomic analysis reveals transcriptional and cell subpopulation differences between human and pig immune cells

Li Jie,Xu Yanan,Zhang Jiayu,Zhang Zhaoqi,Guo Han,Wei Dong,Wu Changhong,Hai Tang,Sun Hai-Xi,Zhao Yong 한국유전학회 2024 Genes & Genomics Vol.46 No.3

Background The pig is a promising donor candidate for xenotransplantation. Understanding the differences between human and swine immune systems is critical for addressing xenotransplant rejection and hematopoietic reconstitution. The gene transcriptional profile differences between human and pig immune cell subpopulations have not been studied. To assess the similarities and differences between pigs and humans at the levels of gene transcriptional profiles or cell subpopulations are important for better understanding the cross-species similarity of humans and pigs, and it would help establish the fundamental principles necessary to genetically engineer donor pigs and improve xenotransplantation. Objective To assess the gene transcriptional similarities and differences between pigs and humans. Methods Two pigs and two healthy humans’ PBMCs were sorted for 10 × genomics single-cell sequence. We generated integrated human-pig scRNA-seq data from human and pig PBMCs and defined the overall gene expression landscape of pig peripheral blood immune cell subpopulations by updating the set of human-porcine homologous genes. The subsets of immune cells were detected by flow cytometry. Results There were significantly less T cells, NK cells and monocytes but more B cells in pig peripheral blood than those in human peripheral blood. High oxidative phosphorylation, HIF-1, glycolysis, and lysosome-related gene expressions in pig CD14+ monocytes were observed, whereas pig CD14+ monocytes exhibited lower levels of cytokine receptors and JAK-STAT-related genes. Pig activated CD4+T cells decreased cell adhesion and inflammation, while enriched for migration and activation processes. Porcine GNLY+CD8+T cells reduced cytotoxicity and increased proliferation compared with human GNLY+CD8+T cells. Pig CD2+CD8+γδT cells were functionally homologous to human CD2+CD4+ γδT cells. Pig CD2−CD8−γδT cells expressed genes with quiescent and precursor characteristics, while CD2−CD8+γδT cells expressed migration and memory-related molecules. Pig CD24+ and CD5+B cells are associated with inflammatory responses. Conclusion Our research with integrated scRNA-seq assays identified the different distribution of pig immune cell subpopulations and the different transcriptional profiles of human and pig immune cells. This study enables a deeper understanding of the development and function of porcine immune cells. Background The pig is a promising donor candidate for xenotransplantation. Understanding the differences between human and swine immune systems is critical for addressing xenotransplant rejection and hematopoietic reconstitution. The gene transcriptional profile differences between human and pig immune cell subpopulations have not been studied. To assess the similarities and differences between pigs and humans at the levels of gene transcriptional profiles or cell subpopulations are important for better understanding the cross-species similarity of humans and pigs, and it would help establish the fundamental principles necessary to genetically engineer donor pigs and improve xenotransplantation. Objective To assess the gene transcriptional similarities and differences between pigs and humans. Methods Two pigs and two healthy humans’ PBMCs were sorted for 10 × genomics single-cell sequence. We generated integrated human-pig scRNA-seq data from human and pig PBMCs and defined the overall gene expression landscape of pig peripheral blood immune cell subpopulations by updating the set of human-porcine homologous genes. The subsets of immune cells were detected by flow cytometry. Results There were significantly less T cells, NK cells and monocytes but more B cells in pig peripheral blood than those in human peripheral blood. High oxidative phosphorylation, HIF-1, glycolysis, and lysosome-related gene expressions in pig CD14+ monocytes were observed, whereas pig CD14+ monocytes exhibited lower levels of cytokine receptors and JAK-STAT-related genes. Pig activated CD4+T cells decreased cell adhesion and inflammation, while enriched for migration and activation processes. Porcine GNLY+CD8+T cells reduced cytotoxicity and increased proliferation compared with human GNLY+CD8+T cells. Pig CD2+CD8+γδT cells were functionally homologous to human CD2+CD4+ γδT cells. Pig CD2−CD8−γδT cells expressed genes with quiescent and precursor characteristics, while CD2−CD8+γδT cells expressed migration and memory-related molecules. Pig CD24+ and CD5+B cells are associated with inflammatory responses. Conclusion Our research with integrated scRNA-seq assays identified the different distribution of pig immune cell subpopulations and the different transcriptional profiles of human and pig immune cells. This study enables a deeper understanding of the development and function of porcine immune cells.

Differential Expression of Th1- and Th2- Type Cytokines in Peripheral Blood Mononuclear Cells of Murrah Buffalo (Bubalus Bubalis) on TLR2 Induction by B. Subtilis Peptidoglycan

Shah, Syed M.,Ravi Kumar, G.V.P.P.S.,Brah, G.S.,Santra, Lakshman,Pawar, Hitesh Asian Australasian Association of Animal Productio 2012 Animal Bioscience Vol.25 No.7

Peripheral blood mononuclear cells (PBMCs) discriminate microbial pathogens and induce T-cell responses of appropriate effector phenotype accordingly. Toll-like receptors (TLRs), in part, mediate this microbial recognition and differentiation while the development of T-cell effector functions critically depends on the release of Th1- or Th2- type cytokines. In the present study, buffalo PBMCs were stimulated under in vitro culture conditions by Bacillus subtilis cell wall petidoglycan, a TLR2 ligand, in a dose- and time- dependent manner. The expression of TLR2 as well as the subsequent differential induction of the Th1 and Th2 type cytokines was measured. Stimulation was analyzed across five doses of peptidoglycan ($10{\mu}g/ml$, $20{\mu}g/ml$, $30{\mu}g/ml$, $40{\mu}g/ml$ and $50{\mu}g/ml$) for 3 h, 12 h, 24 h and 36 h incubation periods. We observed the induction of TLR2 expression in a dose- and time-dependent manner and the peptidoglycan induced tolerance beyond $30{\mu}g/ml$ dose at all incubation periods. The correlation between peptidoglycan stimulation and TLR2 induction was found positive at all doses and for all incubation periods. Increased production of all the cytokines was observed at low doses for 3 h incubation, but the expression of IL-4 was relatively higher than IL-12 at the higher antigen doses, indicating tailoring towards Th2 response. At 12 h incubation, there was a pronounced decrease in IL-4 and IL-10 expression relative to IL-12 in a dose- dependent manner, indicating skewing to Th1 polarization. The expression of IL-12 was highest for all doses across all the incubation intervals at 24 h incubation, indicating Th1 polarization. The relative expression of TNF-${\alpha}$ and IFN-${\gamma}$ was also higher while that of IL-4 and IL-10 showed a decrease. For 36 h incubation, at low doses, relative increase in the expression of IL-4 and IL-10 was observed which decreased at higher doses, as did the expression of all other cytokines. The exhaustion of cytokine production at 36 h indicated that PBMCs became refractory to further stimulation. It can be concluded from this study that the cytokine response to sPGN initially was of Th2 type which skews, more pronouncedly, to Th1 type with time till the cells become refractory to further stimulation.

Effect of Surfactin on Growth Performance of Weaning Piglets in Combination with Bacillus subtilis BC1212

김명석,임종환,박병권,황윤환,송인배,박승춘,윤효인 한국임상수의학회 2009 한국임상수의학회지 Vol.26 No.2

The aim of this study is to investigate the effects of surfactin in combination with Bacillus subtilis BC1212 isolated from Korean soybean paste, on feed utilization and growth performance during 4 weeks in weaning piglets. Eighteen weaning piglets (Landrace × Yorkshire × Duroc; weighing 7.68±0.97 kg) were divided into control (n=9) and experimental groups (n=9). The treatments included a control group consisting of the basal diet with no additives (control) and an experimental group consisting of the basal diet supplemented with 1 g of surfactin C and 1.0×109 CFU of Bacillus subtilis BC1212/kg feed. Piglets fed Bacillus subtilis BC1212 increased in average daily weight gain and feed efficiency. In comparison with the control group, the fecal Bacillus subtilis were significantly increased and the fecal coliform bacteria were markedly reduced in the experimental group. In addition, Bacillus subtilis BC1212 had excellent acid and bile tolerance. The treatment of surfactin (50 μg ml-1) in lipopolysaccharide (LPS)-stimulated swine peripheral blood mononuclear cells (PBMCs) for 6 h showed a significant inhibitory effect on INF-γ, TNF-α and NO secretion (p<0.05) in comparison with LPS treatment alone but not on IL-10 secretion, with levels of secreted IL-10 similar to those secreted by PBMCs stimulated with LPS alone. Supplementation with surfactin in combination with Bacillus subtilis BC1212 in diets improved the ecosystem of gastrointestinal tract by increasing probiotic population and enhanced the systemic immune response in weaned piglets.

급성 골수성 백혈병 환자의 말초혈액 단핵구로부터 수지상세포로의 분화 유도

손상희,이대희,박재선,어완규 고신대학교(의대) 고신대학교 의과대학 학술지 2002 고신대학교 의과대학 학술지 Vol.17 No.1

Background : Dendritic Cells (DCs) are the most potent naturally occuring antigen presenting cells and play an important role in T-cell activation. DCs may be suited for in vivo immunotherapy for its capability to stimulate naive T cell. Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. Methods : In this study, peripheral blood mononuclear cells (PBMCs) from 5 patients with acute myeloid leukemia (AML) were cultured with combinations of Flt-3 Ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-a (TNF-a), and development to DCs was assessed. Peripheral blood mononuclear cells (PBMCs) were obtained from 40-60 ml of peripheral blood of patients with acute myeloid leukemia (AML) by Ficolling. Cells were resuspended in X VIVO-20 medium supplemeted with FL (100ng/ml), GM-CSF (100ng/ml), IL-4 (50ng/ml), and TNF-a (20ng/ml) and seeded into T75 culture flasks at 3x10^7/50ml. Results : After 12 days in culture, cells from 5 samples exhibited morphological and immunophenotypic features of DCs, including expression of CD1a, CD83, and CD86. Conclusion : This study indicates that cells with enhanced antigen-presenting ability can be generated from PBMCs of patients with acute myeloid leukemia, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.

급성 골수성 백혈병 환자의 말초혈액 단핵구로부터 수지상세포로의 분화 유도

손상희,이대희,박재선,어완규 고신대학교 의학부 2002 高神大學校 醫學部 論文集 Vol.17 No.1

Background Dendritic Cells (DCs) are the most potent naturally occuring antigen presenting cells and play an important role in T-cell activation. DCs may be suited for in vivo immunotherapy for its capability to stimulate naive T cell. Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradiation of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. Methods In this study, peripheral blood mononuclear cells (PBMCs) from 5 patients with acute myeloid leukemia (AML) were cultured with combinations of Flt-3 Ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4(IL-4), and tumor necrosis factor-α(TNF-α), and development to DCs was assessed. Peripheral blood mononuclear cells (PBMCs) were obtained from 40-60㎖ of peripheral blood of patients with acute myeloid leukemia (AML) by Ficolling. Cells were resuspended in X VIVO-20 medium supplemented with FL (100ng/㎖), GM-CSF(100ng/㎖), IL-4(50ng/㎖), and TNF-α(20ng-㎖) and seeded into T75 culture flasks at 3×107/50㎖. Results After 12 days in culture, cells from 5 samples exhibited morphological and immunophenotypic features of DCs, including expression of CDla, CD83, and CD86. Conclusion This study indicates that cells with enhanced antigen-presenting ability can be generated from PBMCs of patients with acute myeloid leukemia, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.