Characterization and anti-tumor activities of polysaccharide isolated from Brassica rapa L. via activation of macrophages through TLR2-and TLR4-Dependent pathways.

We have previously shown the immunostimulatory effects by Nozawana (Brassica rapa L.). In this report, we determined the characteristics of Nozawana polysaccharide (NPS) and evaluated the immunomodulatory effects and anti-tumor activity of NPS mediated by macrophage activation. The molecular weight of NPS was determined by gel filtration chromatography with an average molecular weight of approximately 100.6 kDa. HPLC analysis showed that NPS contained glucose, galacturonic acid, galactose, and arabinose. NPS increased cytokine and nitric oxide (NO) production by macrophages in a Toll-like receptor (TLR)2 and TLR4-dependent manner. Furthermore, NPS induced apoptosis significantly against 4T1 murine breast cancer cells cultured in conditioned medium from NPS-treated macrophages through tumor necrosis factor-α. In tumor-bearing mouse model, tumor growth was significantly reduced in NPS-treated mice compared with control mice. These results support the potential use of NPS as an immunotherapeutic material found in health food products.

Heat-killed Lactiplantibacillus plantarum Shinshu N-07 exerts antiobesity effects in western diet-induced obese mice.

AIMS: The aim of this study was to evaluate the antiobesity effects of heat-killed Lactiplantibacillus plantarum Shinshu N-07 (N-07) isolated from fermented Brassica rapa L. METHODS AND RESULTS: Male mice were divided into three groups (n = 10/group); normal diet, western diet (WD), or WD + N-07 (N-07) group and administered each diet for 56 days. The N-07 group showed significant suppression of body weight gain and epididymal fat, perirenal fat, and liver weights compared with the WD group. Higher levels of fecal total cholesterol, triglyceride (TG), and free fatty acid (FFA) were observed in the N-07 group than in the WD group. The mRNA expression of the cholesterol transporter ATP-binding cassette transporter G5 (ABCG5) was significantly increased in the small intestine of N-07-fed mice compared with WD-fed mice. Moreover, N-07 supplementation significantly increased the mRNA expression of ABCG5 and ABCG8 in Caco-2 cells. Furthermore, the TG- and FFA-removal ability of N-07 was confirmed to evaluate its soybean oil- and oleic acid-binding capacities in in vitro experiments. CONCLUSIONS: The antiobesity effects of N-07 might be due to its ability to promote lipid excretion by regulating cholesterol transporter expression and lipid-binding ability.

Modulation of immune function and changes in intestinal flora by Brassica rapa L. (Nozawana).

Dietary foods have functions that can prevent disease and improve immune function, eg. increasing resistance to infection and preventing allergies. Brassica rapa L., known as Nozawana in Japan, is a cruciferous plant and a traditional vegetable of the Shinshu area. Nozawana leaves and stalks are processed mainly into pickled products called Nozawana-zuke. However, it is unclear whether Nozawana exerts beneficial effects on immune function. In this review, we discuss the evidence we have accumulated, which indicate Nozawana has effects on immunomodulation and gut microbiota. We have shown that Nozawana exerts an immunostimulatory effect by enhancing interferon-gamma production and natural killer activity. During the fermentation of Nozawana, the number of lactic acid bacteria increases and cytokine production by spleen cells is enhanced. Moreover, the consumption of Nozawana pickle was shown to modulate gut microbiota and improve the intestinal environment. Thus, Nozawana could be a promising food for improving human health.

Brassica rapa L. prevents Western diet-induced obesity in C57BL/6 mice through its binding capacity of cholesterol and fat.

Obesity, a chronic disorder caused by excessive energy intake leading to fat accumulation in adipose tissue, increases the risk of severe diseases. Brassica rapa L. is known as a traditional vegetable in the Nagano area of Japan. C57BL/6 mice were randomly assigned to three groups, with different diets as follows: a normal diet, a Western diet (WD), and a WD plus B. rapa L. powder (BP) in a 56-day experiment. Brassica rapa L. supplementation reduced the body weight gain and lipid accumulation of mice significantly. The BP group also had higher fecal bile acid, total cholesterol, and triglyceride excretion levels compared with those in the other groups. The antiobesity effects of B. rapa L. were due to its binding with cholesterol and fat, and possibly enhancing the bile acid excretion and modulating gut microbiota, suggesting that B. rapa L. could be a functional vegetable with potential uses in targeting obesity.

Brassica rapa L. activates macrophages via Toll-like receptors.

Macrophages can initiate innate immune responses against microbes and cancer. The aim of this study was to elucidate the effects of Brassica rapa L. on macrophages. The production of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon-γ induced by the insoluble fraction of B. rapa L. was decreased in macrophage-depleted spleen cells compared with controls. The insoluble fraction of B. rapa L. induced expression of H-2Kb, I-Ab, CD40, and CD86, production of cytokines and nitric oxide, and phagocytic activity in RAW264 cells. After treatment with the insoluble fraction, IL-6 and TNF-α production was significantly decreased by anti-Toll-like receptor (TLR)2 mAb or polymyxin B compared with the control. Furthermore, insoluble fraction-mediated cytokine production was significantly lower in peritoneal macrophages from TLR2-/- and TLR4-/- mice compared with wild-type mice. These results suggest that B. rapa L. is a potentially effective immunomodulator for activating macrophages to prevent infections.

Transcriptome analysis of the effect of C-C chemokine receptor 5 deficiency on cell response to Toxoplasma gondii in brain cells.

BACKGROUND: Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain. RESULTS: We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway. CONCLUSIONS: Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.

Enhanced Anti-Allergic Activity of Milk Casein Phosphopeptide by Additional Phosphorylation in Ovalbumin-Sensitized Mice.

The proteolytic digest of milk casein, known as casein phosphopeptide (CPP-III), exhibits diverse biological activities, including calcium absorption and antioxidant activities. We hypothesized that the additional phosphorylation of this peptide can enhance its immunomodulatory activity such as suppression of allergy-associated cytokine and antigen-specific immune response. This study was conducted to assess whether oral intake of additionally phosphorylated CPP-III (P-CPP) attenuates ovalbumin (OVA)-induced IgE-mediated allergic reactions because of the additional phosphate groups. Female BALB/c mice were intraperitoneally sensitized with OVA twice at intervals of 14 days and then orally fed native CPP-III (N-CPP), P-CPP, and dephosphorylated CPP-III (D-CPP) for 6 weeks. Next, the mice were orally challenged with 50 mg of OVA. Oral administration of P-CPP suppressed total and specific IgE levels in the serum. Mice fed P-CPP exhibited low levels of OVA-specific IgG1 and increased OVA-specific IgG2a. P-CPP also suppressed IL-4 production, while D-CPP showed similar a level compared to that of the control. Further, P-CPP increased the population of the T follicular helper (Tfh) cell in the spleen. These results suggest that additional phosphorylation of CPP can enhance the attenuation of allergen-specific IgE-modulated allergic reactions in a murine food allergy model.

Enhancement of natural killer activity and IFN-γ production in an IL-12-dependent manner by a Brassica rapa L.

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity.

Relationship of Enhanced Butyrate Production by Colonic Butyrate-Producing Bacteria to Immunomodulatory Effects in Normal Mice Fed an Insoluble Fraction of Brassica rapa L.

This study was performed to determine the effects of feeding a fiber-rich fraction of Brassica vegetables on the immune response through changes in enteric bacteria and short-chain fatty acid (SCFA) production in normal mice. The boiled-water-insoluble fraction of Brassica rapa L. (nozawana), which consists mainly of dietary fiber, was chosen as a test material. A total of 31 male C57BL/6J mice were divided into two groups and housed in a specific-pathogen-free facility. The animals were fed either a control diet or the control diet plus the insoluble B. rapa L. fraction for 2 weeks and sacrificed to determine microbiological and SCFA profiles in lower-gut samples and immunological molecules. rRNA-based quantification indicated that the relative population of Bacteroidetes was markedly lower in the colon samples of the insoluble B. rapa L. fraction-fed group than that in the controls. Populations of the Eubacterium rectale group and Faecalibacterium prausnitzii, both of which are representative butyrate-producing bacteria, doubled after 2 weeks of fraction intake, accompanying a marginal increase in the proportion of colonic butyrate. In addition, feeding with the fraction significantly increased levels of the anti-inflammatory cytokine interleukin-10 (IL-10) and tended to increase splenic regulatory T cell numbers but significantly reduced the population of cells expressing activation markers. We demonstrated that inclusion of the boiled-water-insoluble fraction of B. rapa L. can alter the composition of the gut microbiota to decrease the numbers of Bacteroidetes and to increase the numbers of butyrate-producing bacteria, either of which may be involved in the observed shift in the production of splenic IL-10.

Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.

Here, we report a simple and low-cost oral oligodeoxynucleotide (ODN) delivery system targeted to the gut Peyer's patches (PPs). This system requires only Dulbecco's modified eagle's medium, calcium chloride, ODNs, and basic laboratory equipment. ODN nanocapsules (ODNcaps) were directly delivered to the PPs through oral administration and were taken up by macrophages in the PPs, where they induced an immune response. Long-term continuous oral dosing with inhibitory/suppressive ODNcaps (iODNcaps, "iSG3caps" in this study) was evaluated using an atopic dermatitis mouse model to visually monitor disease course. Administration of iSG3caps improved skin lesions and decreased epidermal thickness. Underlying this effect is the ability of iSG3 to bind to and prevent phosphorylation of signal transducer and activator of transcription 6, thereby blocking the interleukin-4 signaling cascade mediated by binding of allergens to type 2 helper T cells. The results of our iSG3cap oral delivery experiments suggest that iSG3 may be useful for treating allergic diseases.

Overproduction of Toxoplasma gondii cyclophilin-18 regulates host cell migration and enhances parasite dissemination in a CCR5-independent manner.

BACKGROUND: Toxoplasma gondii hijacks host cells to allow it to disseminate throughout a host animal; however, the migratory machinery involved in this process has not been well characterized. We examined the functional role of T. gondii cyclophilin 18 (TgCyp18) in host cell recruitment using recombinant parasites transfected with TgCyp18. RESULTS: High levels of TgCyp18 enhanced IL-12 production in cysteine-cysteine chemokine receptor 5 (CCR5) knockout mice (CCR5-/-) that had been infected peritoneally with T. gondii. Recruitment of CD11b+ cells to the infection site was enhanced in a CCR5-independent manner. T. gondii spread to several organs, particularly the liver, in a TgCyp18-dependent and CCR5-independent manner. Additionally, CCL5 levels were upregulated in macrophages treated with recombinant protein TgCyp18 and in the peritoneal fluids of the infected CCR5-/- mice. Furthermore, the chemokines involved in macrophage migration, CCL2 and CXCL10, were upregulated in the livers of CCR5-/- mice infected with recombinant parasites that had been transfected with TgCyp18. CONCLUSION: TgCyp18 may play a crucial role in macrophage migration, and in assisting with transport of T. gondii via CCR5-independent mechanisms. TgCyp18 may also play a role with CCL5 in the migration of macrophages to the site of infection, and with CCL2 and CXCL10 in the transport of T. gondii-infected cells to the liver.

Plasmodium berghei circumsporozoite protein encapsulated in oligomannose-coated liposomes confers protection against sporozoite infection in mice.

BACKGROUND: The design and development of an effective malaria vaccine against the pre-erythrocytic and erythrocytic-stages of infection present a great challenge. METHODS: In the present study, protective efficacy of oligomannose-coated liposome (OML)-entrapped merozoite and sporozoite antigens against Plasmodium berghei challenge infection in BALB/c mice was evaluated. RESULTS: Subcutaneous immunization with truncated merozoite surface protein 1 entrapped with OML (OML-PbMSP1) prolonged survival, but failed to protect the mice from erythrocytic-stage infection, despite the antigen-specific antibody responses induced by the immunization regimen. In contrast, immunization with circumsporozoite protein entrapped with OML (OML-PbCSP) elicited antigen-specific humoral and cellular responses, which correlated with substantial protection against sporozoite challenge infections. CONCLUSIONS: The current results represent the use of an oligomannose-coated liposome-based vaccine against pre-erythrocytic and erythrocytic stages malaria infection. This approach may offer a new vaccination strategy against malaria infection.

Mechanisms of interleukin-10 induction in murine spleen and RAW264 cells by Latilactobacillus curvatus K4G4 isolated from fermented Brassica rapa L.

Lactic acid bacteria (LAB) are commonly used in fermented foods, and some LAB modulate the immune response. We aimed to investigate the mechanism by which LAB isolates from fermented Brassica rapa L. induce the production of anti-inflammatory interleukin (IL)-10 by the murine spleen and RAW264 cells. Spleen cells from BALB/c mice or the mouse macrophage cell line RAW264 were cultured with heat-killed LAB isolated from fermented B. rapa L., and the IL-10 level in the supernatant was measured. Latilactobacillus curvatus K4G4 provided the most potent IL-10 induction among 13 isolates. Cell wall components of K4G4 failed to induce IL-10, while treatment of the bacteria with RNase A under a high salt concentration altered K4G4 induction of IL-10 by spleen cells. In general, a low salt concentration diminished the IL-10 induction by all strains, including K4G4. In addition, chloroquine pretreatment and knock down of toll-like receptor 7 through small interfering RNA suppressed K4G4 induction of IL-10 production by RAW264 cells. Our results suggest that single-stranded RNA from K4G4 is involved, via endosomal toll-like receptor 7, in the induction of IL-10 production by macrophages. K4G4 is a promising candidate probiotic strain that modulates the immune response by inducing IL-10 from macrophages.

Correction: Transcriptional profiling of Toll-like receptor 2-deficient primary murine brain cells during Toxoplasma gondii infection.

[This corrects the article DOI: 10.1371/journal.pone.0187703.].

Transcriptome analysis of mouse brain infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.

Effects of one hour daily outdoor access on lying and sleeping postures, and immune traits of tethered cows.

OBJECTIVE: We investigated the effects of outdoor access for 1 h per day on the animal welfare (AW) of tethered cows, in terms of lying and sleeping postures, and immune function. METHODS: A total of five dry cows were tethered all day indoors (tethering) for 30 days and then tethered indoors with 1 h daily outdoor access (ODA-1h) for 30 days. To analyze the effects of ODA-1h, we calculated the total duration and bout frequency per day, and bout duration of lying and sleeping postures during the last five days of each treatment period. We also analyzed the populations of T cells, B cells, and NK cells in peripheral blood mononuclear cells (PBMC) by fluorescence-activated cell sorting and determined the concanavalin A (Con A) -induced proliferation rate of T cells. RESULTS: The mean total time per day of lying during the ODA-1h treatment was significantly shorter than that during the tethering treatment (p<0.001). The Con A-induced proliferation rate of T cells during the ODA-1h treatment was significantly higher than that during the tethering treatment (p = 0.007). The proportion of NK cells in PBMC during the ODA-1h treatment tended to be higher than that during the tethering treatment (p = 0.062). CONCLUSION: Although ODA-1h may decrease lying time, it increases the available space for tethered cows towards that typically found in grazing and free barn feeding systems. This increased available space promotes the expression of normal behaviors such as walking and social behaviors except lying and may also improve the immune function of tethered dry cows, thereby improving their overall welfare.

Procyanidin B2 3,3″-di-O-gallate suppresses IFN-γ production in murine CD4+ T cells through the regulation of glutamine influx via direct interaction with ASCT2.

Excessive activation of CD4+ T cells increases cytokine production substantially and induces immune-mediated diseases. Procyanidins are polyphenols with anti-inflammatory properties. Procyanidin B2 (PCB2) gallate [specifically, PCB2 3,3''-di-O-gallate (PCB2DG)] inhibits cytokine production through the suppression of glycolysis via mammalian target of rapamycin (mTOR) in T cells. Several amino acids play critical roles in T cell activation, especially glutamine, which is important in mTOR signaling and interferon-γ (IFN-γ) production in CD4+ T cells. However, the mechanisms underlying the effects of PCB2DG, including its interaction partners, have yet to be clarified. In the present study, the mechanisms underlying the inhibitory effect of PCB2DG on IFN-γ through glutamine metabolism regulation were investigated. We found that PCB2DG treatment reduced intracellular glutamine levels in CD4+ T cells, whereas the addition of glutamine abrogated the inhibitory effects of PCB2DG on IFN-γ production. The PCB2DG-induced reduction in intracellular glutamine accumulation led to the upregulated expression of activating transcription factor 4, which was induced by the cytoprotective signaling pathway in the amino acid response. In addition, the mRNA and protein expression levels of alanine serine cysteine transporter 2 (ASCT2), a major glutamine transporter in CD4+ T cells, were not altered by PCB2DG treatment. Further analysis using a target identification strategy revealed that PCB2DG binds to ASCT2, suggesting that PCB2DG interacts directly with this major glutamine transporter to inhibit glutamine influx. Overall, this study indicates that ASCT2 is a novel target protein of a dietary polyphenol and provides new insights into the mechanism underlying the immunomodulatory effects of polyphenols.

Procyanidin B2 3,3''-di-O-gallate ameliorates imiquimod-induced skin inflammation by suppressing TLR7 signaling through the inhibition of endosomal acidification in dendritic cells.

The excessive activation of abnormal T helper 17 (Th17) cells and dendritic cells (DCs) in the dermis and epidermis causes severe inflammation of the skin. Toll-like receptor 7 (TLR7)-located in the endosomes of DCs-recognizes nucleic acids from pathogens as well as imiquimod (IMQ), which plays a crucial role in the pathogenesis of skin inflammation. Procyanidin B2 3,3''-di-O-gallate (PCB2DG), a polyphenol, has been reported to suppress the excessive production of proinflammatory cytokines from T cells. The aim of this study was to demonstrate the inhibitory effect of PCB2DG on skin inflammation and TLR7 signaling in DCs. In vivo studies showed that the clinical symptoms of dermatitis were markedly improved by the oral administration of PCB2DG in mouse dermatitis model caused by IMQ application, accompanied by the suppression of excessive cytokine secretion in the inflamed skin and spleen. In vitro, PCB2DG significantly decreased cytokine production in TLR7- or TLR9 ligand-stimulated bone marrow-derived dendritic cells (BMDCs), suggesting that PCB2DG suppresses endosomal toll-like receptors (TLR) signaling in DCs. The activity of endosomal TLRs depends on endosomal acidification, which was significantly inhibited by PCB2DG in BMDCs. The addition of cAMP, an accelerator of endosomal acidification, abrogated the inhibitory effect of cytokine production by PCB2DG. These results provide a new insight into developing functional foods, including PCB2DG, to improve the symptoms of skin inflammation through the suppression of TLR7 signaling in DCs.

Oral administration of procyanidin B2 3,3"-di-O-gallate ameliorates experimental autoimmune encephalomyelitis through immunosuppressive effects on CD4+ T cells by regulating glycolysis.

Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system caused by the excessive activation of T cells. Procyanidins are polyphenols that exhibit anti-inflammatory activity. Procyanidin B2 (PCB2) gallate [specifically, PCB2 3,3″-di-O-gallate (PCB2DG)] inhibits cytokine production in T cells by suppressing the acceleration of glycolysis. In this study, we determined the effect of PCB2DG on T cell-mediated autoimmune disease in vivo. We examined the immunosuppressive effects of PCB2DG using an experimental autoimmune encephalomyelitis (EAE) model, which is a classic animal model for MS. Our results indicated that the clinical score for EAE symptoms improved significantly following the oral administration of PCB2DG. This effect was associated with the suppression of T cell-mediated cytokines (e.g., IFN-γ, TNF-α, and IL-17) and infiltrating T cells into the spinal cord, which ameliorated spinal cord injury. In addition, spleen cell culture experiments revealed that the increase of T cell-mediated pro-inflammatory cytokines in EAE mice was significantly decreased following PCB2DG treatment. We further analyzed the glycolytic activity of spleen cells to identify the mechanism of the immunosuppressive effects of PCB2DG. The production of lactate and the expression of glycolytic enzymes and transporters were increased following EAE induction, but not in PCB2DG-treated EAE mice. Collectively, our results indicate that a dietary polyphenol, which has a unique structure, improves the onset of EAE symptoms and inhibits the excessive activation of T cells by influencing glycolysis.

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner.

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.