• YAKHAK HOEJI
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• v.51 no.4
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• pp.270-276
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• 2007

Silybin is known to be a major active flavonoid component isolated from Silybum marianum, a hepatoprotective medicinal plant. In this study, we examined the immunomodulatory role of silybin on T cell and macrophage-mediated immune responses. To do this, the proliferation of splenic lymphocytes and CD8+ CTLL-2 cells under mitogenic stimulation with lipopolysaccharide (LPS), concanavalin (Con) A and interleukin (IL)-2 and the production of $TNF-{\alpha}$ and NO from LPS- and $IFN-{\gamma}$-activated macrophages was evaluated under silybin treatment. The mitogenic proliferation of splenic lymphocytes induced by LPS and Con A was strongly diminished by silybin in a dose-dependent manner. Moreover, the proliferation of CD8+ CTLL-2 cells was also negatively modulated by the compound. In contrast, silybin did not strongly suppress the proliferation of normal splenocytes and T cell line Sup-T1 cells, indicating that the inhibitory effect of silybin may be due to blocking only mitogenic responses of splenic lymphocytes. In addition, silybin inhibited $TNF-{\alpha}$ production in LPS-stimulated RAW264.7 cells. Effect of silybin however was distinct, according to NO-inducing stimuli. Thus, silybin only blocked NO production induced by $IFN-{\gamma}$ but not LPS and the inhibition was increased when PMA was co-treated with $IFN-{\gamma}$. Unlike NO inhibition, however, this compound protected the cytotoxic damage of RAW264.7 cells induced by both LPS and $IFN-{\gamma}$. Therefore, our data suggest that silybin may participate in host immune responses mediated by T cells and macrophages via regulating mitogenic proliferation, and the production of $TNF-{\alpha}$ and NO, depending on cellular stimuli.

• Biomedical Science Letters
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• v.12 no.1
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• pp.17-22
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• 2006

The baculovirus expression system is a powerful method for producing large amounts of the human erythrocyte-type glucose transport protein, heterologously. Characterization of the expressed protein is expected to show its ability to transport sugars directly. To achieve this, it is a prerequisite to know the properties of the endogenous sugar transport system in Spodoptera frugiperda Clone 21 (Sf21) cells, which are commonly employed as a host permissive cell line to support the baculovirus replication. The Sf21 cells can grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transport system. However, unlike the human glucose transport protein that has a broad substrate and inhibitor specificity, very little is known about the nature of the endogenous sugar transport system in Sf21 cells. In order to characterize further the inhibitor recognition properties of the Sf21 cell transporter, the ability of phloretin, cytochalasin B and D-fructose to inhibit 2-deoxy-D-glucose (2dGlc) transport was examined by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. The 2dGlc transport in the Sf21 cells was very potently inhibited by phloretin, the aglucone of phlorizin with a $K_i$ similar to the value of about $2{\mu}M$ reported for inhibition of glucose transport in human erythrocytes. However, the Sf21 cell transport system was found to differ from the human transport protein in being much less sensitive to inhibition by cytochalasin B (apparent $K_i$ approximately $10\;{\mu}M$). In contrast, It is reported that the inhibitor binds the human erythrocyte counterpart with a $K_d$ of approximately $0.12\;{\mu}M$. Interestingly, the Sf21 glucose transport system also appeared to have high affinity for D-fructose with a $K_i$ of approximately 5mM, contrasting the reported $K_m$ of the human erythrocyte transport protein for the ketose of 1.5M.

• International Journal of Oral Biology
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• v.40 no.1
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• pp.11-17
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• 2015

The gingival epithelium of the oral cavity is constantly exposed to exogenous stimuli such as bacterial toxins, allergens, and thermal changes. These exogenous stimuli are resisted by innate host defense in gingival epithelial cells. However, it is unclear exactly how the exogenous stimuli affect detrimentally on the human gingival epithelial cells. Here, we investigated whether the allergen, such as house dust mite (HDM) extract, is linked to $Ca^{2+}$ signaling and proinflammatory cytokine expression in primary cultured human gingival epithelial cells. HDM extract induced an increase in intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in a dose-dependent manner. Extracellular $Ca^{2+}$ depletion did not affected on the HDM extract-induced increase in $[Ca^{2+}]_i$. The HDM extract-induced increase in $[Ca^{2+}]_i$ was abolished by the treatment with U73122 and 2-APB, which are inhibitors of phospholipase C (PLC) and inositol 1,4,5-trisphosphate ($IP_3$) receptor. Moreover, HDM extract induced the mRNA expression of pro-inflammatory cytokine, interleukin (IL)-8. These results suggest that HDM extract triggers $PLC/IP_3$-dependent $Ca^{2+}$ signaling and IL-8 mRNA expression in primary cultured human gingival epithelial cells.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.78-78
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• 2003

Inulin, an active component of Chicorium intybus root, has been shown to stimulate the growth of bifidobacteria, and inhibit colon carcinogenesis. NO mediates a number of the host-defense functions of activated macrophages, including antimicrobial and tumoricidal activity. We examined the effect of inulin on the synthesis of NO in RAW 264.7 cells. Inulin alone had no effect, whereas inulin with IFN-ν synergistically increased the NO production and inducible NO synthase (iNOS) expression in RAW 264.7 cells. Synergy between IFN-ν and inulin was mainly dependent on inulin-induced TNF-${\alpha}$ secretion. Also, protein kinase C (PKC)-${\alpha}$ was involved in the inulin-induced NO production. Inulin-mediated NO production was inhibited by the protein tyrosine kinase (PTK) inhibitor, tyrphostin AG126. Since iNOS gene transcriptions have been shown to be under the control of the NF -$\kappa$B/Rel family of transcription factors, we assessed the effect of inulin on NF -$\kappa$B/Rel using an EMSA. Inulin produced strong induction of NF-$\kappa$B/Rel binding, whereas AP-l binding was slightly induced in RAW 264.7 cells. Inulin stimulated phosphorylation and degradation of I$\kappa$B-${\alpha}$. These results suggest that in IFN-ν-primed RAW 264.7 cells inulin might stimulate NO synthesis via activation of PKC-${\alpha}$ and PTK, resulting in the activation of NF-$\kappa$B.

• Restorative Dentistry and Endodontics
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• v.25 no.4
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• pp.606-618
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• 2000

Dental pulp infection is most commonly caused by extensive dental caries, and some bacterial species invade root canals; bacterial components and products are thought to be associated with the pathogenesis of periapical periodontitis. A principle driving force behind pulpal disease response appears to lie in the host immune system's to bacteria and their products. We examined the production of interleukin $1{\beta}$ (IL-$1{\beta}$) and tumor necrosis factor ${\alpha}$(TNF-${\alpha}$) from human peripheral mononuclear cells, lymphocytes and monocytes stimulated by heat-killed Acitnobacillus actinomycetemcomitans (ATCC 29523), Porphyromonas gingivalis (ATCC 33277) and Prevotella intermedia (ATCC 25611), and also by their sonicated bacterial extracts (SBE), respectively. The effects of three strains of heat-killed bacteria and their SBEs on the morphology of cultured blood cell lines HL-60 (KCLB 10240) and J774A.1 (KCLB 40067) were observed under the inverted microscope. Ultrastructural changes of J774A.1 exposed to heat-killed P. intermedia and its SBE were investigated using transmission electron microscopy. Production of IL-$1{\beta}$ was reduced in human peripheral mononuclear cells after stimulation by sonic bacterial extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. Heat-killed and sonic extract of P. gingivalis inhibited the production of TNF-${\alpha}$ in peripheral mononuclear cells. Production of TNF-${\alpha}$ was inhibited in peripheral monocytes after stimulation by sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. HL-60 and J 774A.1 cells showed granular degeneration after treatment with heat-killed and sonic extracts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia Chromatin margination and shrinkage were observed in 774A.1 treated with heat-killed P. intermedia. Cell wall structure and organelles were destroyed and vacuoles were formed in cytoplasm in J774A.1 treated with P. intermedia sonic extract. These results suggest that A actinomycetemcomitans, P gingivalis and P intermedia may have an important role in the formation and progression of pulpal diseases via both modulation of production of IL-$1{\beta}$ and TNF-${\alpha}$ from blood mononuclear cells and cytopathic effects.

• Archives of Plastic Surgery
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• v.35 no.4
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• pp.367-372
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• 2008

Purpose: Prevention of acute rejection in skin allografts without continuous immunosuppression lacks reports in worldwide literature. Needs for chronic immunosuppression preclude the use of tissue allograft as a routine surgical reconstructive option. Recently dendritic cells(DC) gained considerable attention as antigen presenting cells that are also capable of immunologic tolerance induction. This study assesses the effects of alloantigen-pulsed dendritic cells in induction of survival increase in a rat skin allograft model. Methods: Recipient-derived dendritic cells were harvested from rat whole blood and cultured with GM-CSF(200 ng/mL) and IL-4(8 ng/mL) for 2 weeks. Then donor-specific alloantigen pulsed dendritic cells were reinjected into tail vein before skin graft. The rat dorsal skin allografts were transplanted in 5 subgroups. Groups: I) untreated, II) anti-lymphocyte serum(ALS, 0.5 mL), III) FK-506(2 mg/kg), IV) DCp, VI) DCp and FK-506. Graft appearance challenges were assessed postoperatively. Results: The group V(DC and FK-506 treated) showed longest graft survival rate(23.5 days) than other groups; untreated(5.8 days), ALS(7.2 days), FK-506 (17.5 days), DCp(12.2 days). Conclusion: Donor antigen pulsed host dendritic cell combined with short-term immunosuppression prolong skin allograft survival and has potential therapeutic application for induction of donor antigen specific tolerance.

• IMMUNE NETWORK
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• v.11 no.1
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• pp.68-78
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• 2011

Background: Graft-versus-host disease (GVHD) is a huddle for success of hematopoietic stem cell transplantation. In this study, effects of irradiation dose on immune kinetics of GVHD were investigated using B6 ${\rightarrow}$ BALB.B system, a mouse model for GVHD after MHC-matched allogeneic transplantation. Methods: BALB.B mice were transplanted with bone marrow and spleen cells from C57BL/6 mice after irradiation with different doses. Leukocytes residing in the peripheral blood and target organs were collected periodically from the GVHD hosts for analysis of chimerism formation and immune kinetics along the GVHD development via flow cytometry. Myeloid cells were tested for production of IL-17 via flow cytometry. Results: Pre-conditioning of BALB.B hosts with 900 cGy and 400 cGy resulted in different chimerism of leukocytes from the blood and affected survival of GVHD hosts. Profiles of leukocytes infiltrating GVHD target organs, rather than profiles of peripheral blood leukocytes (PBLs), were significantly influenced by irradiation dose. Proportions of IL-17 producing cells in the infiltrating $Gr-1^+$ or $Mac-1^+$ cells were higher in the GVHD hosts with high does irradiation than those with low dose irradiation. Conclusion: Pre-conditioning dose affected tissue infiltration of leukocytes and cytokine production by myeloid cells in the target organs.

• International Journal of Oral Biology
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• v.36 no.2
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• pp.91-101
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• 2011

MspTL is the major surface protein of Treponema lecithinolyticum associated with periodontitis and endodontic infections. Our recent investigation revealed that MspTL induces proinflammatory cytokines and intercellular adhesion molecule 1 in THP-1 cells and periodontal ligament cells. In this study we conducted oligonucleotide microarray analysis to investigate the global transcriptional regulation in THP-1 cells stimulated with purified recombinant MspTL. MspTL upregulated the expression of 90 genes in THP-1 cells at least four fold, and the functions of these genes were categorized into adhesion, apoptosis/antiapoptosis, cell cycle/growth/differentiation, chemotaxis, cytoskeleton organization, immune response, molecular metabolism, proteolysis, signaling, and transcription. The majority of the modified genes are known to be NF-${\kappa}B$-responsive and interferon-stimulated genes (ISGs). The expression of 12 selected genes was confirmed by real-time RT-PCR. Because prostaglandin $E_2(PGE_2)$ is an important inflammatory mediator and Cox-2 was found to be induced by MspTL in the microarray analysis, we determined the level of $PGE_2$ in the culture supernatants of MspTL-treated cells and found that MspTL significantly increased $PGE_2$. Our results provide insight into the gene regulation of host cells in response to MspTL, and may contribute to the understanding of the molecular mechanism in periodontitis.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.679-691
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• 1993

As a series of studies to investigate the effect of immunosuppression on Ascaris suum infection in undefinitive hosts, a delicate relationship between host and parasite, rabbits were divided into experiment 1(control group) and experiment 2(immnunosuppressive group treated with prednisolone acetate) and inoculated with a single dose of 5,000 embryonated A suum eggs. The recovery rates, sizes and morphology of the larvae and immunological responses in the rabbits were chronologically monitored according to somatic migration. In both experiments, the larvae failed to develop into the adults, but young adults in the experiment 2 grew somewhat faster and survied later than those in the experiment 1. The mast cells of small intestinal mucosa and mesenteric lymph nodes and the goblet cells of small intestinal mucosa in the worm detected cases of experiment 2 decreased remarkably in number comparing with those of experiment 1. Considering the experimental results. the expulsion mechanism of somatic migrant larvae may he related to the temporary increasing tendency of the mast cells, the goblet cells, T-cells of mesenteric lymph nodes and spleens, eosinophils in peripheral blood, degranulation rates of peritoneal mast cells and the migration inhibition rates of leucocytes. In addition, patent infection of A suum in the rabbits was not obviously observed despite of immunosuppression by prednisolone acetate.

• Animal Bioscience
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• v.34 no.3_spc
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• pp.463-470
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• 2021

Objective: This experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP. Methods: Nitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit. Results: P700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 μM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700. Conclusion: In animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.