Anti-Inflammatory Effects of Spiramycin in LPS-Activated RAW 264.7 Macrophages.

Drug repurposing is a simple concept with a long history, and is a paradigm shift that can significantly reduce the costs and accelerate the process of bringing a new small-molecule drug into clinical practice. We attempted to uncover a new application of spiramycin, an old medication that was classically prescribed for toxoplasmosis and various other soft-tissue infections; specifically, we initiated a study on the anti-inflammatory capacity of spiramycin. For this purpose, we used murine macrophage RAW 264.7 as a model for this experiment and investigated the anti-inflammatory effects of spiramycin by inhibiting the production of pro-inflammatory mediators and cytokines. In the present study, we demonstrated that spiramycin significantly decreased nitric oxide (NO), interleukin (IL)-1ß, and IL-6 levels in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Spiramycin also inhibited the expression of NO synthase (iNOS), potentially explaining the spiramycin-induced decrease in NO production. In addition, spiramycin inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs); extracellular signal-regulated kinase (ERK) and c-Jun N terminal kinase (JNK) as well as the inactivation and subsequent nuclear translocation of nuclear factor κB (NF-κB). This indicated that spiramycin attenuates macrophages' secretion of IL-6, IL-1ß, and NO, inducing iNOS expression via the inhibition of the NF-κB and MAPK signaling pathways. Finally, we tested the potential application of spiramycin as a topical material by human skin primary irritation tests. It was performed on the normal skin (upper back) of 31 volunteers to determine whether 100 µM and µM of spiramycin had irritation or sensitization potential. In these assays, spiramycin did not induce any adverse reactions. In conclusion, our results demonstrate that spiramycin can effectively attenuate the activation of macrophages, suggesting that spiramycin could be a potential candidate for drug repositioning as a topical anti-inflammatory agent.

Anti-melanogenic effects of hot-water extracts from Torreya nucifera via MAPKs and cAMP signaling pathway on B16F10 cells.

Torreya nucifera is an evergreen tree in the family Taxaceae, the seeds, leaves, and stems of which have long been used as edible products and herbal medicines in Korea. Previous studies of biological activity have shown that T. nucifera has antioxidant and anti-inflammatory effects. However, the effect of T. nucifera leaves on melanogenesis are yet to be studied. In this investigation, we used B16F10 melanoma cells to test the efficacy of T. nucifera leaf hot water extract (TLWE). α-melanocyte stimulating hormone (α-MSH) stimulated B16F10 melanoma cells were treated with various concentrations of TLWE (50, 100, and 200 µg/mL). The results showed that TLWE reduced the melanin content and cellular tyrosinase activity in a concentration-dependent manner. It also inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) in the mitogen-activated protein kinase (MAPK) signaling pathway. The compounds catechin and ρ-coumaric acid, which are known to have a whitening effect on skin, were detected by HPLC analysis. These results suggest that TLWE has an anti-melanogenic effect. In addition, the safety of TLWE was tested. The results of the skin irritation test showed that TLWE is harmless to the human skin, even at higher concentrations than those used in the experiment. Therefore, we suggest that the water extract of T. nucifera leaves has potential for use as a skin-whitening agent.

Modern Virtual Fencing Application: Monitoring and Controlling Behavior of Goats Using GPS Collars and Warning Signals.

This paper describes our virtual fence system for goats. The present invention is a method of controlling goats without visible physical fences and monitoring their condition. Control occurs through affecting goats, using one or more sound signals and electric shocks when they attempt to enter a restricted zone. One of the best Machine Learning (ML) classifications named Support Vector Machines (SVM) is used to observe the condition. A virtual fence boundary can be of any geometrical shape. A smart collar on goats' necks can be detected by using a virtual fence application. Each smart collar consists of a global positioning system (GPS), an XBee communication module, an mp3 player, and an electrical shocker. Stimuli and classification results are presented from on-farm experiments with a goat equipped with smart collar. Using the proposed stimuli methods, we showed that the probability of a goat receiving an electrical stimulus following an audio cue (dog and emergency sounds) was low (20%) and declined over the testing period. Besides, the RBF kernel-based SVM classification model classified lying behavior with an extremely high classification accuracy (F-score of 1), whilst grazing, running, walking, and standing behaviors were also classified with a high accuracy (F-score of 0.95, 0.97, 0.81, and 0.8, respectively).

Antimelanogenic Effects of Polygonum tinctorium Flower Extract from Traditional Jeju Fermentation via Upregulation of Extracellular Signal-Regulated Kinase and Protein Kinase B Activation.

This study was carried out to investigate the antimelanogenic effects of a Polygonum tinctorium flower extract obtained using red nuruk, a traditional Jeju barley-based fermentation starter. We also studied the mechanism of action of the P. tinctorium fermented flower extract (PTFFE) in mouse melanoma cells (B16F10). Cells were treated with various concentrations (62.5, 125 and 250 µg/mL) of PTFFE and the results showed that PTFFE significantly decreased the melanin content and tyrosinase activity without being cytotoxic. In addition, PTFFE strongly inhibited the expression of tyrosinase and tyrosinase-related protein 2 by decreasing the expression of the microphthalmia-associated transcription factor, as shown by a western blot assay. Furthermore, PTFFE inhibited melanogenesis via upregulation of the phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B, also known as AKT. We also used inhibitors such as PD98059 (a specific ERK inhibitor) or LY294002 (an AKT inhibitor) to determine whether the signaling pathways are involved. High-performance liquid chromatography fingerprinting showed the presence of a quercetin glucoside (isoquercitrin) and quercetin in PTFFE. To test the potential for PTFFE application as a cosmetic material, we also performed a primary skin irritation test on human skin. In this assay, PTFFE did not induce any adverse reactions at the treatment dose. Based on these results, we suggest that PTFFE may be considered a potential antimelanogenesis candidate for topical applications.

Password-only authenticated three-party key exchange with provable security in the standard model.

Protocols for password-only authenticated key exchange (PAKE) in the three-party setting allow two clients registered with the same authentication server to derive a common secret key from their individual password shared with the server. Existing three-party PAKE protocols were proven secure under the assumption of the existence of random oracles or in a model that does not consider insider attacks. Therefore, these protocols may turn out to be insecure when the random oracle is instantiated with a particular hash function or an insider attack is mounted against the partner client. The contribution of this paper is to present the first three-party PAKE protocol whose security is proven without any idealized assumptions in a model that captures insider attacks. The proof model we use is a variant of the indistinguishability-based model of Bellare, Pointcheval, and Rogaway (2000), which is one of the most widely accepted models for security analysis of password-based key exchange protocols. We demonstrated that our protocol achieves not only the typical indistinguishability-based security of session keys but also the password security against undetectable online dictionary attacks.

Protein kinase C δ (PKCδ)-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition-mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)-induced endotoxemia.

Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)-induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation under LPS-stressed conditions via protein kinase C δ (PKCδ) activation. Furthermore, PKCδ-induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS-treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition was restored in an ERK1/2-dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK1/2 activation via PKCδ relieved LPS-mediated endotoxemia. This finding suggests that IL-10 hyperexpression resulting from GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway for endotoxemia.

Resveratrol suppresses tumor progression via the regulation of indoleamine 2,3-dioxygenase.

This study showed the potential of resveratrol to inhibit the expression and activity of interferon-γ (IFN-γ)-induced indoleamine 2,3-dioxygenase (IDO) in bone marrow-derived dendritic cells (BMDCs). The mechanism of suppression was associated with the activity of Janus kinase/signal transducers and activators of transcription (JAK/STAT) and protein kinase Cδ (PKCδ). In addition, resveratrol-mediated IDO suppression in IFN-γ-stimulated BMDCs appears to play a pivotal role in anti-tumor activity through the regulation of CD8(+) T cell polarization and cytotoxic T lymphocyte (CTL) activity. Systemic administration of resveratrol suppressed tumor growth in EG7 thymoma-bearing mice in an IDO-dependent manner. Taken together, resveratrol not only regulates immune response through the regulation of IDO in a JAK/STAT1- and PKCδ-dependent manner, but also modulates the IDO-mediated immune tolerance in EG7 thymoma.

A simple segmentation and quantification method for numerical quantitative analysis of cells and tissues.

BACKGROUND: Microscopic image analysis based on image processing is required for quantitative evaluation of decellularization. Existing methods are not widely used because of expensive commercial software, and machine learning-based techniques lack generality for decellularization because many high-resolution image data has to be processed. OBJECTIVE: In this study, we developed an image processing algorithm for quantitative analysis of tissues and cells in a general microscopic image. METHODS: The proposed method extracts the color images obtained by the microscope into reference images consisting of grayscale, red (R), green (G), and blue (B) information and transforms each into a binary image. The transformed images were extracted by separating the cells and tissues through outlier noise elimination, logical multiplication and labeling. In order to verify the method, decellularization of porcine arotic valve was performed by the electrical method. Slice samples were obtained by time and the proposed method was applied. RESULTS: The experimental results show that the segmentation of cells and tissues, and quantitative analysis of the number of cells and changes in tissue area during the decellularization process was possible. CONCLUSIONS: The proposed method shows that cell and tissue extraction and quantitative numerical analysis were possible in different brightness of microscopic images.

Induction of myeloma-specific cytotoxic T lymphocytes ex vivo by CD40-activated B cells loaded with myeloma tumor antigens.

We investigated to establish CD40-activated B cells (CD40-B cells) as alternative antigen-presenting cells (APCs) for the induction of myeloma-specific cytotoxic T lymphocytes (CTLs). To generate CD40-B cells, peripheral blood mononuclear cells were co-cultured with CD40L-transfected J558 cells in the presence of IL-4, insulin, transferrin, and cyclosporine for 14 days, and pulsed with myeloma lysates. The CD40-B cells consistently expressed high levels of CD80, CD86, CD54, CCR7, and HLA-DR. The CD40-B cells produced IL-12, IFN-gamma, and IL-6 during the culture period, but not IL-10. In addition, the CD40-B cells showed potent allogeneic T-cell stimulatory capacities that depended on the dose ratio and had the potential to polarize naïve T cells into Th1 subsets. The CD40-B cells loaded with tumor lysates induced strong target-specific CTLs, based on large numbers of IFN-gamma secreting cells and higher cytotoxic activity against target cells compared to the CD40-B cells without the tumor lysates. These results suggest that CD40-B cells loaded with myeloma lysates might provide alternative APCs for cellular immunotherapy in patients with myeloma.

Monocyte-derived dendritic cells from HLA-matched allogeneic donors showed a greater ability to induce leukemic cell-specific T cells in comparison to leukemic cell-derived dendritic cells or monocyte-derived dendritic cells from AML patients.

We investigated the usefulness of allogeneic monocyte-derived dendritic cells (allogeneic mDCs) pulsed with leukemic lysates in acute myeloid leukemia (AML). Allogeneic mDCs showed higher expressions of several molecules (HLA-DR, CD80, CD83 or CD86), higher production of IL-12 and higher capacity to stimulate allogeneic T cells compared to both leukemic DCs and autologous mDCs. Autologous T cells primed by allogeneic mDCs displayed a larger number of interferon-gamma-secreting cells against leukemic cells than those primed by either leukemic DCs or autologous mDCs. These results suggest that monocyte-derived DCs from HLA-matched allogeneic donors can be used as an alternative to generate leukemia-specific cytotoxic T cells and to overcome the limitation of leukemic DCs or autologous mDCs.

Induction of multiple myeloma-specific cytotoxic T lymphocyte stimulation by dendritic cell pulsing with purified and optimized myeloma cell lysates.

We investigated the possibility of immunotherapy for multiple myeloma (MM) using myeloma-specific cytotoxic T lymphocytes (CTLs) that were stimulated in vitro by dendritic cells (DCs) pulsing with purified and optimized myeloma lysates. CD14(+) cells were cultured in the presence of GM-CSF and IL-4. On day 6, the immature DCs were pulsed with the purified myeloma cell lysates, and then maturation of the DCs was induced by the addition of a cytokine cocktail. There were no differences in the phenotypic expressions of mature DCs that were generated by pulsing with CD138(+) cell lysates or total cell lysates. In optimization of the concentration of myeloma lysates, DCs pulsed with 10 microg/mL of myeloma lysate had greater allogeneic T-cell stimulatory capacities than those pulsed with higher concentrations of myeloma lysates. The CTL lines generated by DCs pulsed with myeloma lysates demonstrated potent cytotoxic activities against autologous target cells, but not against HLA-A2(-) cell lines or K562 cell lines. The DCs pulsed with myeloma lysates demonstrated a higher stimulatory capacity for autologous CTL compared with mature nonpulsed DCs. These results suggested that the DCs pulsed with purified and optimized myeloma cell lysates could generate potent myeloma-specific CTLs for approaches in MM.

Toxoplasma gondii-derived heat shock protein 70 stimulates maturation of murine bone marrow-derived dendritic cells via Toll-like receptor 4.

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) induced maturation of bone marrow-derived dendritic cells (DCs) of wild-type (WT) C57BL/6 mice as evidenced by an increase in surface expression of MHC class I and II molecules and costimulatory molecules such as CD40, CD80, and CD86. Functionally, decreased phagocytic ability and increased alloreactive T cell stimulatory ability were observed in T.g.HSP70-stimulated DCs. These phenotypic and functional changes of T.g.HSP70-stimulated DCs were demonstrated in Toll-like receptor (TLR) 2- and myeloid differentiation factor 88 (MyD88)-deficient but not TLR4-deficient C57BL/6 mice. DCs from WT and TLR2-deficient but not TLR4-deficient mice produced IL-12 after T.g.HSP70 stimulation. T.g.HSP70-stimulated DCs from WT, TLR2-deficient, and MyD88-deficient, but not TLR4-deficient mice expressed IFN-beta mRNA. Thus, T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade.

Down-regulation of cellular vascular endothelial growth factor levels induces differentiation of leukemic cells to functional leukemic-dendritic cells in acute myeloid leukemia.

We examined the effect of cellular vascular endothelial growth factor (VEGF) levels on the generation of leukemic dendritic cells (DCs). Leukemic DCs were successfully generated in vitro from bone marrow cells of 16 of 21 acute myeloid leukemia (AML) patients, and the cellular VEGF concentrations in the leukemic cells and the neutralization of VEGF with anti-VEGF antibody were determined. AML cells that failed to generate leukemic DCs showed significantly higher cellular VEGF levels compared with generated leukemic DCs, and down-regulation of cellular VEGF levels induced the generation of leukemic DCs from AML cells. Inhibition of cellular VEGF levels increased interleukin (IL)-12 production and the allostimulatory capacity of leukemic DCs. These results suggest that the generation of leukemic DCs from AML cells is inversely related to the VEGF production of the cells and that the down-regulation of cellular VEGF levels can induce potential differentiation of leukemic cells to functional leukemic DCs in patients with AML.

Heat shock protein X purified from Mycobacterium tuberculosis enhances the efficacy of dendritic cells-based immunotherapy for the treatment of allergic asthma.

Dendritic cells play an important role in determining whether naïve T cells mature into either Th1 or Th2 cells. We determined whether heat-shock protein X (HspX) purified from Mycobacterium tuberculosis regulates the Th1/Th2 immune response in an ovalbumin (OVA)-induced murine model of asthma. HspX increased interferon-gamma, IL-17A, -12 and transforming growth factor (TGF)-ß production and T-bet gene expression but reduced IL-13 production and GATA-3 gene expression. HspX also inhibited asthmatic reactions as demonstrated by an increase in the number of eosinophils in bronchoalveolar lavage fluid, inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyper-responsiveness. Furthermore, HspX enhanced OVA-induced decrease of regulatory T cells in the mediastinal lymph nodes. This study provides evidence that HspX plays critical roles in the amelioration of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of HspX with respect to its effects on a murine model of asthma.

Stimulation of early gene induction and cell proliferation by lysophosphatidic acid in human amnion-derived WISH cells: role of phospholipase D-mediated pathway.

Human-amniotic WISH cells express the lysophosphatidic acid (LPA) receptor, LPA(1), LPA(2) but not LPA(3). When WISH cells were stimulated with LPA, phospholipase D (PLD) activation was dramatically induced via a cytosolic calcium increase and protein kinase C activation. We also found that LPA stimulated two kinds of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and p38 kinase via PLD-dependent signaling pathways in WISH cells. In terms of the LPA-mediated functional modulation of WISH cells, we observed that LPA stimulates the induction of two early genes (c-Jun and c-Fos) and cellular proliferation in WISH cells. We examined the signaling pathways involved in LPA-mediated cellular responses. LPA-induced early gene induction was completely blocked by normal butanol (n-butanol) but not by t-butanol, suggesting that PLD activity is essentially required for the process. PD98059 (2'-amino-3'-methoxyflavone) but not SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) also significantly blocked LPA-induced early gene induction, suggesting a crucial role for ERK. Pertussis toxin (PTX) did not affect on the LPA-induced early gene induction and ERK activation, ruling out the role of Gi/o protein(s) in the process. The cellular proliferation of WISH cells was also dramatically inhibited by n-butanol or PD98059. This study demonstrates the physiological role of LPA on the modulation of early gene induction and on WISH cell proliferation, and the crucial role played by PLD in the process.

Toxoplasma gondii-derived heat shock protein HSP70 functions as a B cell mitogen.

We have investigated the role of Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) as a B cell mitogen by measuring proliferative responses in vitro. TgHSP70 induced prominent proliferative responses in murine B cells derived not only from T gondii-infected but also from uninfected mice. Nude mice responded to TgHSP70; however, severe combined immunodeficiency, RAG1-/- B6, and microMT mice failed to respond. B220+ spleen cells showed marked proliferation after stimulation with TgHSP70, but neither CD4+ nor CD8+ population responded. This unresponsiveness of CD4+ and CD8- T cells to TgHSP70 was antigen presenting cells independent. These data indicate that TgHSP70 induced the proliferation of B cells but not T cells. Polymyxin B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate TgHSP70-induced proliferation. C3H/HeN mice responded well to TgHSP70 stimulation; however, C3H/HeJ mice carrying a point mutation in the Toll-like receptor (TLR) 4 failed to respond. This indicates that TLR4 is required for TgHSP70-induced B cell activation. The involvement of TLR4 in the TgHSP70-induced proliferative responses of spleen cells was also shown by the use of TLR4-/- mice. But TgHSP70-induced, but not LPS-induced, spleen cell proliferation was observed in MyD88-/- mice, indicating that the MyD88 molecule was involved in LPS-induced proliferation but not in TgHSP70-induced proliferation.

Trp-Lys-Tyr-Met-Val-Met stimulates phagocytosis via phospho-lipase D-dependent signaling in mouse dendritic cells.

Dendritic cells (DCs) play a key role in activating the immune response against invading pathogens as well as dying cells or tumors. Although the immune response can be initiated by the phagocytic activity by DCs, the molecular mechanism involved in this process has not been fully investigated. Trp-Lys-Tyr-Met-Val-Met-NH(2) (WKYMVM) stimulates the activation of phospholipase D (PLD) via Ca(2+) increase and protein kinase C activation in mouse DC cell line, DC2.4. WKYMVM stimulates the phagocytic activity, which is inhibited in the presence of N-butanol but not t-butanol in DC2.4 cells. Furthermore, the addition of phosphatidic acid, an enzymatic product of PLD activity, enhanced the phagocytic activity in DC2.4 cells. Since at least two of formyl peptide receptor (FPR) family (FPR1 and FPR2) are expressed in DC2.4 as well as in mouse bone marrow-derived dendritic cells, this study suggests that the activation of FPR family by WKYMVM stimulates the PLD activity resulting in phagocytic activity in DC2.4 cells.

Up-regulation of phospholipase Cgamma1 and phospholipase D during the differentiation of human monocytes to dendritic cells.

Phospholipase C (PLC)gamma and phospholipase D (PLD) play pivotal roles in the signal transduction required for various cellular responses, including cell proliferation and differentiation. Dendritic cells (DCs), which are professional antigen-presenting cells, can be generated from human monocytes by stimulating the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). We investigated whether PLCgamma and PLD expression levels can be changed during the differentiation of the human monocytes into DCs. The enzymatic activity and protein level of PLC gamma1 were significantly increased in the human monocyte-derived DCs by GM-CSF/IL-4, but the protein levels of PLC gamma2 were unaltered. Moreover, the enzymatic activity and protein level of PLD1b and PLD2 were up-regulated during the differentiation of human monocytes to DCs, but those of PLD1a were not changed. A higher phagocytic activity of DCs was found to be correlated with the up-regulations of PLCgamma1 and PLD, and the phagocytic activity of DCs was inhibited by a PLC-specific inhibitor (U73122) and by a phosphatidic acid acceptor (n-butanol), but to be increased by phosphatidic acid. Thus, suggesting that PLC and PLD participate in the process. This study suggests that the up-regulations of PLCgamma1 and PLD are accompanied by the differentiation of monocytes into DCs, which results in increased phagocytic activity.

Activation of formyl peptide receptor-like 1 by WKYMVm induces serine phosphorylation of STAT3, which inhibits its tyrosine phosphorylation and nuclear translocation induced by hydrogen peroxide.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that modulates the expression of several genes. The activation of STAT3 accompanies tyrosine phosphorylation and its translocation to the nucleus. Formyl peptide receptor like 1 (FPRL1) is an important classical chemoattractant receptor. In this study, we observed that the stimulation of FPRL1 by Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) caused serine phosphorylation but not tyrosine phosphorylation of STAT3 in a pertussis toxin-sensitive manner. Moreover, downstream of FPRL1 stimulation, phospholipase D (PLD) activity was dramatically increased. n-butanol, a well-known phosphatidic acid (PA) acceptor, completely inhibited WKYMVm-induced STAT3 serine phosphorylation. Moreover, the exogenous addition of PA mimicked STAT3 phosphorylation by WKYMVm. We also found that WKYMVm stimulated extracellular signal regulated kinase (ERK), and that ERK activity is required for STAT3 serine phosphorylation. This WKYMVm-induced ERK activation was inhibited by n-butanol, whereas ERK activation was also induced by the addition of exogenous PA. In terms of the functional aspects of the WKYMVm-induced serine phosphorylation of STAT3, we found that hydrogen peroxide-stimulated STAT3 activation was blocked by pretreating WKYMVm. Taken together, we found that WKYMVm stimulated FPRL1, and that this resulted in STAT3 serine phosphorylation via PLD-mediated ERK activation, and that the serine phosphorylation of STAT3 blocked hydrogen peroxide-induced STAT3 activity.

Rhamnogalacturonan II is a Toll-like receptor 4 agonist that inhibits tumor growth by activating dendritic cell-mediated CD8+ T cells.

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1ß, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.