Immunostimulatory potential of extruded plant-based meat: effect of extrusion moisture level on macrophage activation.

In this study, the effect of different moisture levels in extruded plant-based meat on macrophage immunostimulation, and the potential of this meat as a protein source and a solution to environmental and economic challenges associated with conventional meat was investigated. To determine the effects of the extruded plant-based meat, cell viability assay, enzyme-linked immunosorbent assay, flow cytometry, and western blotting were performed. Low-moisture (LMME) and high-moisture meat extracts (HMME) showed higher potential to activate macrophages and regulate cytokine production than raw material extract. Treatment with LMME and HMME resulted in increased expression of CD80, CD86, and MHC class I/II proteins, indicating their potential to activate macrophages. Western blotting suggested that the immune activation observed in a previous study of macrophages was because of the phosphorylation of MAPKs and NF-κB. These findings suggest that extruded plant-based meat can potentially be used as an immunostimulatory food ingredient.

CM1, a Chrysin Derivative, Protects from Endotoxin-Induced Lethal Shock by Regulating the Excessive Activation of Inflammatory Responses.

Sepsis, a leading cause of death worldwide, is a harmful inflammatory condition that is primarily caused by an endotoxin released by Gram-negative bacteria. Effective targeted therapeutic strategies for sepsis are lacking. In this study, using an in vitro and in vivo mouse model, we demonstrated that CM1, a derivative of the natural polyphenol chrysin, exerts an anti-inflammatory effect by inducing the expression of the ubiquitin-editing protein TNFAIP3 and the NAD-dependent deacetylase sirtuin 1 (SIRT1). Interestingly, CM1 attenuated the Toll-like receptor 4 (TLR4)-induced production of inflammatory cytokines by inhibiting the extracellular-signal-regulated kinase (ERK)/MAPK and nuclear factor kappa B (NF-κB) signalling pathways. In addition, CM1 induced the expression of TNFAIP3 and SIRT1 on TLR4-stimulated primary macrophages; however, the anti-inflammatory effect of CM1 was abolished by the siRNA-mediated silencing of TNFAPI3 or by the genetic or pharmacologic inhibition of SIRT1. Importantly, intravenous administration of CM1 resulted in decreased susceptibility to endotoxin-induced sepsis, thereby attenuating the production of pro-inflammatory cytokines and neutrophil infiltration into the lung compared to control mice. Collectively, these findings demonstrate that CM1 has therapeutic potential for diverse inflammatory diseases, including sepsis.

Protective Effects of Bombyx batryticatus Protein-Rich Extract Against Cisplatin-Induced Nephrotoxicity in HEK293 Cells and a Mouse Model.

Cisplatin, a potent and prominent chemotherapeutic drug, has considerable side effects, including nephrotoxicity, which limits its therapeutic application and efficacy. Therefore, the development of agents that protect normal cells while preserving cisplatin's chemotherapeutic properties is of utmost importance. This study aimed to explore the protective effects of Bombyx batryticatus protein-rich extract (BBPE) against cisplatin-induced nephrotoxicity in a cisplatin-treated mouse model and human embryonic kidney (HEK293) cells. Apoptosis was assessed in HEK293 cells to determine the cytoprotective effects of BBPE and its effects on the generation of cisplatin-induced reactive oxygen species (ROS) and mitochondrial transmembrane potential (MTP) collapse. Although cisplatin induced nephrotoxicity in HEK293 cells, pretreatment with BBPE showed significant protective effects against cisplatin-induced nephrotoxicity by regulating the expression levels of pro- and antiapoptotic proteins. The cytoprotective effects of BBPE were mediated by decreased ROS production and MTP loss in cisplatin-treated HEK293 cells. The in vitro results were confirmed in the cisplatin-treated mouse model. Pretreatment with BBPE protected against cisplatin-induced nephrotoxicity by restoring malondialdehyde, superoxide dismutase, and catalase levels in kidney tissue and blood urea nitrogen and creatinine serum levels. Furthermore, histopathological assessment and terminal dUTP nick end-labeling staining showed that BBPE mitigated cisplatin-induced nephrotoxicity in kidney tissues. Overall, BBPE may act as a potent agent for alleviating cisplatin-induced nephrotoxicity, thereby increasing the safety of cisplatin-based chemotherapy.

Deinococcus radiodurans-derived membrane vesicles protect HaCaT cells against H2O2-induced oxidative stress via modulation of MAPK and Nrf2/ARE pathways.

BACKGROUND: Deinococcus radiodurans is a robust bacterium that can withstand harsh environments that cause oxidative stress to macromolecules due to its cellular structure and physiological functions. Cells release extracellular vesicles for intercellular communication and the transfer of biological information; their payload reflects the status of the source cells. Yet, the biological role and mechanism of Deinococcus radiodurans-derived extracellular vesicles remain unclear. AIM: This study investigated the protective effects of membrane vesicles derived from D. radiodurans (R1-MVs) against H2O2-induced oxidative stress in HaCaT cells. RESULTS: R1-MVs were identified as 322 nm spherical molecules. Pretreatment with R1-MVs inhibited H2O2-mediated apoptosis in HaCaT cells by suppressing the loss of mitochondrial membrane potential and reactive oxygen species (ROS) production. R1-MVs increased the superoxide dismutase (SOD) and catalase (CAT) activities, restored glutathione (GSH) homeostasis, and reduced malondialdehyde (MDA) production in H2O2-exposed HaCaT cells. Moreover, the protective effect of R1-MVs against H2O2-induced oxidative stress in HaCaT cells was dependent on the downregulation of mitogen-activated protein kinase (MAPK) phosphorylation and the upregulation of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. Furthermore, the weaker protective capabilities of R1-MVs derived from ΔDR2577 mutant than that of the wild-type R1-MVs confirmed our inferences and indicated that SlpA protein plays a crucial role in R1-MVs against H2O2-induced oxidative stress. CONCLUSION: Taken together, R1-MVs exert significant protective effects against H2O2-induced oxidative stress in keratinocytes and have the potential to be applied in radiation-induced oxidative stress models.

Low-dose radiation therapy suppresses viral pneumonia by enhancing broad-spectrum anti-inflammatory responses via transforming growth factor-ß production.

Low-dose radiation therapy (LDRT) can suppress intractable inflammation, such as that in rheumatoid arthritis, and is used for treating more than 10,000 rheumatoid arthritis patients annually in Europe. Several recent clinical trials have reported that LDRT can effectively reduce the severity of coronavirus disease (COVID-19) and other cases of viral pneumonia. However, the therapeutic mechanism of LDRT remains unelucidated. Therefore, in the current study, we aimed to investigate the molecular mechanism underlying immunological alterations in influenza pneumonia after LDRT. Mice were irradiated to the whole lung 1 day post-infection. The changes in levels of inflammatory mediators (cytokines and chemokines) and immune cell populations in the bronchoalveolar lavage (BALF), lungs, and serum were examined. LDRT-treated mice displayed markedly increased survival rates and reduced lung edema and airway and vascular inflammation in the lung; however, the viral titers in the lungs were unaffected. Levels of primary inflammatory cytokines were reduced after LDRT, and transforming growth factor-ß (TGF-ß) levels increased significantly on day 1 following LDRT. Levels of chemokines increased from day 3 following LDRT. Additionally, M2 macrophage polarization or recruitment was increased following LDRT. We found that LDRT-induced TGF-ß reduced the levels of cytokines and polarized M2 cells and blocked immune cell infiltration, including neutrophils, in BALF. LDRT-induced early TGF-ß production was shown to be a key regulator involved in broad-spectrum anti-inflammatory activity in virus-infected lungs. Therefore, LDRT or TGF-ß may be an alternative therapy for viral pneumonia.

Polysaccharides from Annona muricata leaves protect against cisplatin­induced cytotoxicity in macrophages by alleviating mitochondrial dysfunction.

Cisplatin is a prominent chemotherapeutic agent that can induce significant damage to normal cells. Therefore, it is important to develop agents that protect normal cells without influencing the chemotherapeutic effect of cisplatin. The present study was conducted to explore the protective effects of Annona muricata leaf polysaccharides (ALPS) against cisplatin­induced toxicity in macrophages. Apoptosis was assessed in macrophages and lung cancer cells to investigate the cytoprotective effect of ALPS, their effect on the production of cisplatin­induced reactive oxygen species (ROS) and the loss of the mitochondrial transmembrane potential (MTP). Cisplatin, when used alone or in combination with ALPS, showed significant toxicity against A549 and H460 lung cancer cells. However, cisplatin­induced cytotoxicity was suppressed by cotreatment of RAW 264.7 macrophages with ALPS. ALPS significantly inhibited the upregulation of Bax, cytosolic cytochrome c and caspases­3, ­8 and ­9. Moreover, ALPS resulted in the cleavage of PARP and downregulation of Bcl­2 levels in a concentration­dependent manner, which ultimately led to a reduction in the apoptotic and necrotic populations of cisplatin­treated RAW 264.7 macrophages. The suppression of the apoptotic signaling pathways was mediated through the reduction of ROS and MTP loss in cisplatin­treated RAW 264.7 macrophages. In addition, ALPS alleviated cell damage by suppressing the mitochondrial apoptotic pathways in cisplatin­treated bone marrow­derived macrophages. Together, these findings suggested that ALPS may alleviate the toxic side effects of chemotherapeutic agents and act as a potential candidate for use as an effective adjuvant therapy.

Deinococcus radiodurans R1 Lysate Induces Tolerogenic Maturation in Lipopolysaccharide-Stimulated Dendritic Cells and Protects Dextran Sulfate Sodium-Induced Colitis in Mice.

Deinococcus radiodurans is an extremophilic bacterium that can thrive in harsh environments. This property can be attributed to its unique metabolites that possess strong antioxidants and other pharmacological properties. To determine the potential of D. radiodurans R1 lysate (DeinoLys) as a pharmacological candidate for inflammatory bowel disease (IBD), we investigated the anti-inflammatory activity of DeinoLys in bone marrow-derived dendritic cells (BMDCs) and a colitis mice model. Lipopolysaccharide (LPS)-stimulated BMDCs treated with DeinoLys exhibited alterations in their phenotypic and functional properties by changing into tolerogenic DCs, including strongly inhibited proinflammatory cytokines (TNF-α and IL-12p70) and surface molecule expression and activated DC-induced T cell proliferation/activation with high IL-10 production. These phenotypic and functional changes in BMDCs induced by DeinoLys in the presence of LPS were abrogated by IL-10 neutralization. Furthermore, oral administration of DeinoLys significantly reduced clinical symptoms against dextran sulfate sodium-induced colitis, including body weight loss, disease activity index, histological severity in colon tissue, and lower myeloperoxidase level in mice. Our results establish DeinoLys as a potential anti-inflammatory candidate for IBD therapy.

Effects of Gamma Irradiation on Inhibition of Urease Activity and Fishy Smell in Mackerel (Scomber japonicus) during Refrigerated Storage.

In this study, gamma-irradiated mackerel (Scomber japonicus) meat was stored in a refrigerator for 20 days to examine the physicochemical changes related to fishy smell. The effect of gamma irradiation on the inhibition of the activity of crude urease extracted from Vibrio parahaemolyticus was also evaluated. Increased levels of trimethylamine (TMA) and volatile basic nitrogen (VBN) content, which are the main components causing fishy smell, were significantly reduced by day 20 of storage after gamma irradiation, indicating that freshness was maintained during storage. The ammonia nitrogen contents of 3, 7, 10, and 20 kGy gamma-irradiated groups were significantly decreased by 6.5, 15.2, 17.4, and 23.9%, respectively, compared to non-irradiated groups on day 20 of storage. In addition, urease activity decreased in a gamma irradiation intensity-dependent manner. Volatile organic compounds (VOCs) were measured during the storage of gamma-irradiated mackerel meat. The contents of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavors due to spoilage of fish, were significantly reduced by day 20 of storage. Therefore, gamma irradiation can be considered useful for inhibiting urease activity and reducing fishy smell during fish storage.

Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant, Petasites japonicus, via the Induction of Murine Dendritic Cell Maturation.

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8+ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.

Radiation-Inactivated S. gallinarum Vaccine Provides a High Protective Immune Response by Activating Both Humoral and Cellular Immunity.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Protective Effect of Polysaccharides Extracted from Cudrania tricuspidata Fruit against Cisplatin-Induced Cytotoxicity in Macrophages and a Mouse Model.

Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.

Molecular Characteristics of IS1216 Carrying Multidrug Resistance Gene Cluster in Serotype III/Sequence Type 19 Group B Streptococcus.

Streptococcus agalactiae is the leading cause of meningitis in newborns and a significant cause of invasive diseases in pregnant women and adults with underlying diseases. Antibiotic resistance against erythromycin and clindamycin in group B streptococcus (GBS) isolates has been increasing worldwide. GBS expresses the Srr1 and Srr2 proteins, which have important roles in bacterial infection. They have been investigated as novel vaccine candidates against GBS infection, with promising results. But a recent study detected non-srr1/2-expressing clinical isolates belonging to serotype III. Thus, we aimed to analyze the genotypes of non-srr1/2 GBS clinical isolates collected between 2013 and 2016 in South Korea. Forty-one (13.4%) of the 305 serotype III isolates were identified as non-srr1/2 strains, including sequence type 19 (ST19) (n = 16) and ST27 (n = 18) strains. The results of the comparative genomic analysis of the ST19/serotype III/non-srr1/2 strains further revealed four unique gene clusters. Site 4 in the srr1 gene locus was replaced by an lsa(E)-lnu(B)-aadK-aac-aph-aadE-carrying multidrug-resistant gene cluster flanked by two IS1216 transposases with 99% homology to the enterococcal plasmid pKUB3007-1. Despite the Srr1 and Srr2 deficiencies, which resulted in reduced fibrinogen binding, the adherence of non-srr1/2 strains to endothelial and epithelial cells was comparable to that of Srr1- or Srr2-expressing strains. Moreover, their virulence in mouse models of meningitis was not significantly affected. Furthermore, additional adhesin-encoding genes, including a gene encoding a BspA-like protein, which may contribute to colonization by non-srr1/2 strains, were identified via whole-genome analysis. Thus, our study provides important findings that can aid in the development of vaccines and antibiotics against GBS. IMPORTANCE Most previously isolated group B streptococcus (GBS) strains express either the Srr1 or Srr2 glycoprotein, which plays an important role in bacterial colonization and invasion. These glycoproteins are potential protein vaccine candidates. In this study, we first report GBS clinical isolates in which the srr1/2 gene was deleted or replaced with foreign genes. Despite Srr1/2 deficiency, in vitro adherence to mammalian cells and in vivo virulence in murine models were not affected, suggesting that the isolates might have another adherence mechanism that enhanced their virulence aside from Srr1/2-fibrinogen-mediated adherence. In addition, several non-srr1/2 isolates replaced the srr1/2 gene with the lnu(B) and lsa(E) antibiotic resistance genes flanked by IS1216, effectively causing multidrug resistance. Collectively, we believe that our study identifies the underlying genes responsible for the pathogenesis of new GBS serotype III. Furthermore, our study emphasizes the need for alternative antibiotics for patients who are allergic to ß-lactams and for those who are pregnant.

Chrysin Derivative CM1 and Exhibited Anti-Inflammatory Action by Upregulating Toll-Interacting Protein Expression in Lipopolysaccharide-Stimulated RAW264.7 Macrophage Cells.

Although our previous study revealed that gamma-irradiated chrysin enhanced anti-inflammatory activity compared to intact chrysin, it remains unclear whether the chrysin derivative, CM1, produced by gamma irradiation, negatively regulates toll-like receptor (TLR) signaling. In this study, we investigated the molecular basis for the downregulation of TLR4 signal transduction by CM1 in macrophages. We initially determined the appropriate concentration of CM1 and found no cellular toxicity below 2 µg/mL. Upon stimulation with lipopolysaccharide (LPS), CM1 modulated LPS-stimulated inflammatory action by suppressing the release of proinflammatory mediators (cytokines TNF-α and IL-6) and nitric oxide (NO) and downregulated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, CM1 markedly elevated the expression of the TLR negative regulator toll-interacting protein (Tollip) in dose- and time-dependent manners. LPS-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II), proinflammatory cytokines (TNF-α and IL-6), COX-2, and iNOS-mediated NO were inhibited by CM1; these effects were prevented by the knockdown of Tollip expression. Additionally, CM1 did not affect the downregulation of LPS-induced expression of MAPKs and NF-κB signaling in Tollip-downregulated cells. These findings provide insight into effective therapeutic intervention of inflammatory disease by increasing the understanding of the negative regulation of TLR signaling induced by CM1.

Immunoregulatory properties of a crude extraction fraction rich in polysaccharide from Chrysanthemum zawadskii Herbich var. latilobum and its potential role as a vaccine adjuvant.

The objective of the current study was to demonstrate the immunostimulatory effects of a polysaccharide isolated from Chrysanthemum zawadskii Herbich var. latilobum leaves (CP) and evaluate its potential as a vaccine adjuvant. Results showed that CP induced maturation of the dendritic cells (DCs). In addition, CP-treated DCs activated naïve T cells to polarized CD4+ and CD8+ T cells and substantially induced the production of IFN-γ and IL-2 in vitro. Furthermore, CP initiated the maturation of DCs via the activation of MAPK and NF-κB signaling pathways. Interestingly, systemic administration of CP-treated DCs pulsed with ovalbumin (OVA) peptides significantly enhanced the immune response in vivo, which included the generation of antigen (OVA)-specific polyfunctional T cells, increased cytotoxic T lymphocyte activity, induction of Th1-mediated humoral immunity, and suppression of tumor growth. Taken together, our study highlighted the immunoregulatory activity of CP as well as its potential as a candidate vaccine adjuvant.

HMOC, a chrysin derivative, induces tolerogenic properties in lipopolysaccharide-stimulated dendritic cells.

Although we previously identified a new hydroxymethoxyl chrysin derivative (HMOC) using ionizing radiation, the anti-inflammatory mechanism of HMOC in dendritic cells remains unclear. In this study, we investigate the effects of HMOC on phenotypic and functional changes in activated bone marrow-derived dendritic cells (BMDCs). In lipopolysaccharide (LPS)-stimulated BMDCs, HMOC treatment inhibited pro-inflammatory cytokines (TNF-α, IL-12p70, and IL-1ß), surface molecules (CD80, CD86, MHC-I, and MHC-II), and antigen-presentation to MHC-I and II without a decrease in IL-10. Furthermore, HMOC increased indoleamine 2,3-dioxygenase-1 (IDO1) activity via activation of JNK and p38 signaling in the presence of LPS. Interestingly, LPS-stimulated DCs treated with HMOC inhibited the proliferation and activation of CD4+ and CD8+ T cells, as well as differentiation of CD4+ T cells into Th1-, Th2- and Th17 cells. In addition, LPS-stimulated DCs treated with HMOC induced an increase in CD4+CD25+Foxp3+ regulatory T cells (Tregs). Collectively, our results suggest that HMOC confers tolerogenic properties in BMDCs, which are responsible for inducing Th cell differentiation to Tregs. Our findings provide a better understanding of the anti-inflammatory mechanism of HMOC in DCs and may contribute to development of a valuable therapeutic candidate for atopic dermatitis.

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine.

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4+ and CD8+ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4+ and CD8+ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.

Salmonella Vaccine Vector System for Foot-and-Mouth Disease Virus and Evaluation of Its Efficacy with Virus-Like Particles.

Foot-and-mouth disease virus (FMDV) causes a highly contagious and devastating disease in livestock animals and has a great potential to cause severe economic loss worldwide. The major antigen of FMDV capsid protein, VP1, contains the major B-cell epitope responsible for effectively eliciting protective humoral immunity. In this study, irradiated Salmonella Typhimurium (KST0666) were used as transgenic vectors containing stress-inducible plasmid pRECN-VP1 to deliver the VP1 protein from FMDV-type A/WH/CHA/09. Mice were orally inoculated with ATOMASal-L3 harboring pRECN-VP1, and FMDV virus-like particles, where (VLPFMDV)-specific humoral, mucosal, and cellular immune responses were evaluated. Mice vaccinated with attenuated Salmonella (KST0666) expressing VP1 (named KST0669) showed high levels of VLP-specific IgA in feces and IgG in serum, with high FMDV neutralization titer. Moreover, KST0669-vaccinated mice showed increased population of IFN-γ (type 1 T helper cells; Th1 cells)-, IL-5 (Th2 cells)-, and IL-17A (Th17 cells)-expressing CD4+ as well as activated CD8+ T cells (IFN-γ+CD8+ cells), detected by stimulating VLPFMDV. All data indicate that our Salmonella vector system successfully delivered FMDV VP1 to immune cells and that the humoral and cellular efficacy of the vaccine can be easily evaluated using VLPFMDV in a Biosafety Level I (BSL1) laboratory.

Galangin treatment during dendritic cell differentiation confers tolerogenic properties in response to lipopolysaccharide stimulation.

Tolerogenic dendritic cells (tolDCs) can induce the differentiation of immunosuppressive regulatory T cells and are therefore candidates for the prevention or treatment of various inflammatory diseases. Galangin, a major component of propolis and Alpinia officinarum, has well-established anti-inflammatory effects, but its ability to induce a tolerogenic state in DCs has not been demonstrated. In this study, we investigated the effects of galangin on DC differentiation and immune responses. In particular, we compared phenotypic and functional differences between DCs (Gal-DCs) generated by galangin treatment during DC differentiation and bone marrow-derived DCs. Gal-DCs were generated by adding culture medium containing various doses of galangin (1.8-18.5 µM) on 3 and 6 day. Upon lipopolysaccharide (100 ng/mL) stimulation for 24 h, Gal-DCs generated with 7.4 µM galangin treatment showed lower levels of CD86 and lower major histocompatibility complex class II antigen-presentation than those of bone marrow-derived DCs. Furthermore, Gal-DCs showed markedly increased programmed death ligand 1 expression and IL-10 production via the activation of mitogen-activated protein kinases. Interestingly, Gal-DCs co-cultured with allogeneic CD4 T cells exhibited the reduced cell proliferation and differentiation into Th1-, Th2-, and Th17-type cell; instead, Gal-DCs contributed to the induction of CD4+CD25+Foxp3+ Tregs. Taken together, our data suggest that exposure to galangin during DC differentiation confers tolerogenic properties, efficiently inducing Th cell differentiation to immunosuppressive Tregs. These findings provide new insights into the molecular mechanism underlying the anti-inflammatory effects of galangin on DCs.

Comparison of Exosomes Derived from Non- and Gamma-Irradiated Melanoma Cancer Cells as a Potential Antigenic and Immunogenic Source for Dendritic Cell-Based Immunotherapeutic Vaccine.

Cancer cells can secrete exosomes under various stressful conditions, whose functions are involved in the delivery of various biologically active materials into host cells and/or modulation of host immune responses. Therefore, an improved understanding of the immunological interventions that stress-induced tumor exosomes have may provide novel therapeutic approaches and more effective vaccine designs. Here, we confirmed the phenotypical and functional alterations of dendritic cells (DCs), which act as a bridge between the innate and adaptive arms of immunity, following non-irradiated (N-exo) and gamma-irradiated melanoma cancer cell-derived exosome (G-exo) stimulation, and evaluated the N-exo- and G-exo-stimulated DCs as therapeutic cancer vaccine candidates. We demonstrated that G-exo-stimulated DCs result in DC maturation by the upregulation of surface molecule expression, pro-inflammatory cytokine release, and antigen-presenting ability, and the downregulation of endocytic capacity. In addition, these cells promoted T cell proliferation and the generation of T helper type 1 (Th1) and interferon (IFN)-γ-producing CD8+ T cells. However, N-exo-stimulated DCs induced semi-mature phenotypes and functions, eventually inhibiting T cell proliferation, decreasing IFN-γ, and increasing IL-10-producing CD4+ T cells. In addition, although N-exo and G-exo stimulations showed similar levels of antigen-specific IFN-γ production, which served as tumor antigen sources in melanoma-specific T cells, G-exo-stimulated DC vaccination conferred a stronger tumor growth inhibition than N-exo-stimulated DC vaccination; further, this was accompanied by a high frequency of tumor-specific, multifunctional effector T cells. These results suggest that gamma irradiation could provide important clues for designing and developing effective exosome vaccines that can induce strong immunogenicity, especially tumor-specific multifunctional T cell responses.

Promotion of Cellular and Humoral Immunity against Foot-and-Mouth Disease Virus by Immunization with Virus-Like Particles Encapsulated in Monophosphoryl Lipid A and Liposomes.

Virus-like particles (VLPs) have emerged as promising vaccine candidates against foot-and-mouth disease (FMD). However, such vaccines provide a relatively low level of protection against FMD virus (FMDV) because of their poor immunogenicity. Therefore, it is necessary to design effective vaccine strategies that induce more potent immunogenicity. In order to investigate the means to improve FMD VLP vaccine (VLPFMDV) immunogenicity, we encapsulated VLPs (MPL/DDA-VLPFMDV) with cationic liposomes based on dimethyldioctadecylammonium bromide (DDA) and/or monophosphoryl lipid A (MPL, TLR4 agonist) as adjuvants. Unlike inactivated whole-cell vaccines, VLPFMDV were successfully encapsulated in this MPL/DDA system. We found that MPL/DDA-VLPFMDV could induce strong cell-mediated immune responses by inducing not only VLP-specific IFN-γ+CD4+ (Th1), IL-17A+CD4+ (Th17), and IFN-γ+CD8+ (activated CD8 response) T cells, but also the development of VLP-specific multifunctional CD4+ and CD8+ memory T cells co-expressing IFN-γ, TNF-α, and IL-2. In addition, the MPL/DDA-VLPFMDV vaccine markedly induced VLP-specific antibody titers; in particular, the vaccine induced greater Th1-predominant IgG responses than VLPFMDV only and DDA-VLPFMDV. These results are expected to provide important clues for the development of an effective VLPFMDV that can induce cellular and humoral immune responses, and address the limitations seen in current VLP vaccines for various diseases.