Productions of Th2 cytokines, IL-4 and IL-10, were enhanced via the function of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells treated with caffeic acid phenethyl ester.

OBJECTIVES: Interleukin (IL)-2 production by mouse spleen cells stimulated with an anti-CD3 antibody is significantly enhanced by caffeic acid phenethyl ester (CAPE), a major constituent of Chinese propolis (CP). In this study, we evaluated the functional significance of IL-2 in CAPE-treated activated spleen cells. METHODS: Mouse spleen cells were stimulated with an anti-CD3 monoclonal antibody in the presence of CAPE. Cytokine production was examined using an enzyme-linked immunosorbent assay (ELISA). Messenger RNA level expression was examined via reverse transcription quantitative polymerase chain reaction (RT-PCR). IL-2 function was assessed using IL-2 and a neutralizing antibody. Spleen cell subsets were identified and characterized using flow cytometry. RESULTS: CAPE treatment of anti-CD3 antibody-stimulated spleen cells reduced IFN-γ production, then enhanced IL-2 production, followed by enhancement of IL-4 and IL-10 production. The Th2 cytokine production enhancing effects of CAPE were completely abolished by addition of an anti-IL-2 neutralizing antibody. In the absence of CAPE, exogenously added IL-2 could enhance IL-4 production to a lesser degree, but did not stimulate IL-10 production, in stimulated spleen cells. Interestingly, CAPE significantly reduced the proportions of CD4+ and CD8+ cells, and increased those of CD4-CD8- cells among anti-CD3 stimulated spleen cells, in the presence or absence of anti-IL-2 neutralizing antibody treatment. CONCLUSIONS: CAPE reduced IFN-γ production, then enhanced IL-4 and IL-10 production via the activity of specifically elevated IL-2 in stimulated spleen cells. CAPE exerted these effects in a CD4- CD8- cell specific manner.

Bioactivity of human dental pulp-derived stem cells with boron-controlled S-PRG filler eluate by anion exchange.

Surface pre-reacted glass-ionomer (S-PRG) filler is a bioactive glass filler capable of releasing various ions. A culture medium to which was added an S-PRG filler eluate rich in boron was reported to enhance alkaline phosphatase (ALP) activity in human dental pulp-derived stem cells (hDPSC). To clarify the role of boron eluted from S-PRG fillers, the modified S-PRG filler eluate with different boron concentrations was prepared by using an anion exchange material. Therefore, elemental mapping analysis of anion exchange material, adsorption ratio, hDPSCs proliferation and ALP activity were evaluated. For statistical analysis, Kruskal-Wallis test was used, with statistical significance determined at p<0.05. ALP activity enhancement was not observed in hDPSC cultured in the medium that contained the S-PRG filler eluate from which boron had been removed. The result suggested the possibility that an S-PRG filler eluate with controlled boron release could be useful for the development of novel dental materials.

Chemokine­like receptor 1­positive cells are present in the odontoblast layer in tooth tissue in rats and humans.

Cluster of differentiation (CD)44 is a marker of dental pulp stem cells and is involved in odontoblast differentiation and calcification. Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) is also expressed in odontoblasts and dental pulp stem cells and is involved in inflammation suppression and tooth regeneration. Resolvin E1, a bioactive lipid, is a CMKLR1 ligand that mediates the chemerin-CMKLR1 interaction and suppresses pulpal inflammation. The present study clarified the intracellular and tissue localization of CD44 and CMKLR1 by immunohistochemical staining of normal pulp and pulp with pulpitis from 12-week-old male Wistar rat teeth or human teeth. In addition, the localization of CD44 and CMKLR1 in human dental pulp stem cells was observed by immunofluorescence staining. The present study also examined the involvement of resolvin E1 in inhibiting inflammation and calcification by western blotting. CD44- and CMKLR1-positive cells were confirmed in the odontoblast layer in normal dental pulp of rats and humans. CD44 was mainly localized in the cell membrane and CMKLR1 was mainly found in the cytoplasm of human dental pulp stem cells. CMKLR1 was also confirmed in the odontoblast layer in rats and humans with pulpitis but CD44 was not present. Following treatment of dental pulp stem cells with lipoteichoic acid, which imitates Gram-positive bacterial infection, resolvin E1 did not suppress the expression of cyclooxygenase-2 or of the odontoblast differentiation marker, dentin sialophosphoprotein. Furthermore, resolvin E1 induced the differentiation of dental pulp stem cells into odontoblasts even in the presence of the inflammatory stimulus.

Enhanced production of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells by caffeic acid phenethyl ester, a major component of Chinese propolis.

OBJECTIVES: We evaluated the immune-modulatory effects of Chinese propolis (CP) and its major constituent, caffeic acid phenethyl ester (CAPE), on the cytokine production of anti-CD3 antibody-stimulated mouse spleen cells. METHODS: Mouse spleen cells stimulated by anti-CD3 monoclonal antibody were co-cultured with CP, CAPE, and HC030031, a specific antagonist of the TRPA1 Ca2+-permeable cation channel. Cytokine production was assayed by enzyme-linked immunosorbent assay. Interleukin (IL)-2 mRNA expression was examined by reverse transcription-quantitative polymerase chain reaction. RESULTS: In stimulated spleen cells treated with 1/16,000 CP diluent, IL-2 production was markedly enhanced, while IL-4 and IL-10 productions were not significantly affected. In contrast, interferon (IFN)-γ, IL-6, and IL-17 productions were markedly reduced. These effects of CP were reproduced by the CAPE treatment. A time-course observation demonstrated that, compared to control cells, IL-2 mRNA expression and production were significantly enhanced in the spleen cells stimulated by CAPE; however, IL-2 production was markedly delayed compared to that in the untreated control cells. The enhancement of IL-2 production by CAPE was scarcely alleviated by the addition of HC030031. These effects of CAPE upon IL-2 mRNA production were abolished in spleen cells without anti-CD3 antibody stimulation. CONCLUSIONS: CAPE is an important regulator of CP for cytokine regulation in anti-CD3 antibody-stimulated spleen cells. The agent specifically reduced IFN-γ, IL-6, and IL-17 and slightly enhanced Th2 cytokine production while significantly enhancing IL-2 production at the transcriptional level.

The Role of Immune Modulatory Cytokines in the Tumor Microenvironments of Head and Neck Squamous Cell Carcinomas.

HNSCCs are the major progressive malignancy of the upper digestive and respiratory organs. Malignant phenotypes of HNSCCs are regulated by the pro- and anti-tumoral activities of the immune modulatory cytokines associated with TMEs, i.e., a representative pro-inflammatory cytokine, interferon (IFN)-γ, plays a role as an anti-tumor regulator against HNSCCs; however, IFN-γ also drives programmed death-ligand (PD-L) 1 expression to promote cancer stem cells. Interleukin (IL)-2 promotes the cytotoxic activity of T cells and natural killer cells; however, endogenous IL-2 can promote regulatory T cells (Tregs), resulting in the protection of HNSCCs. In this report, we first classified and mentioned the immune modulatory aspects of pro-inflammatory cytokines, pro-/anti-inflammatory cytokines, and anti-inflammatory cytokines upon HNSCC phenotypes. In the TME of HNSCCs, pro-tumoral immune modulation is mediated by stromal cells, including CAFs, MDSCs, pDCs, and TAMs. Therefore, we evaluated the functions of cytokines and chemokines that mediate the crosstalk between tumor cells and stromal cells. In HNSCCs, the status of lymph node metastasis is an important hallmark of a worse prognosis. We therefore evaluated the possibility of chemokines mediating lymph node metastases in HNSCC patients. We also mention therapeutic approaches using anti-tumoral cytokines or immunotherapies that target cytokines, chemokines, or signal molecules essential for the immune evasion of HNSCCs. We finally discuss modulation by HPV infection upon HNSCC phenotypes, as well as the prognostic significance of serum cytokine levels in HNSCC patients.

Enhanced production of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis.

OBJECTIVES: In this report, we attempt to clarify the immune modulatory effects of Brazilian green propolis (BGP) and its major component, artepillin C, on the cytokine production of anti-CD3 antibody-stimulated mouse spleen cells. We also estimate the physiological mechanism affecting artepillin C's upon the cells. METHODS: Male C3H/HeN mouse spleen cells stimulated by antiCD3 monoclonal antibody were co-cultured with BGP, artepillin C, and HC030031, a transient receptor potential ankyrin 1 (TRPA1) Ca2+ channel antagonist. The synthesis of interferon (IFN)-γ, interleukin (IL)-6, IL-17, IL-4, IL-10, and IL-2 was assayed by enzyme-linked immunoassay. The expression of IL-2 mRNA and the protein product were examined by reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively. RESULTS: The production of IL-2 was markedly enhanced, while that of IL-4 and IL-10 was not significantly affected; by contrast, the production of IFN-γ, IL-6, and IL-17 was significantly reduced in the antibody-stimulated spleen cells treated with BGP at a non-cytostatic concentration. These effects were reproduced in the cells treated with artepillin C. The expression of IL-2 mRNA was unaffected; however, that of the protein was significantly enhanced in the artepillin C-treated cells compared to untreated control cells. The enhancement of protein expression and the production of IL-2 by artepillin C was significantly alleviated by adding HC030031. CONCLUSIONS: Artepillin C is an important regulator of cytokine synthesis from activated spleen cells. The agent specifically augmented the expression of IL-2 via the Ca2+-permeable cation channel, TRPA1, at least in part, at the translational or secretion levels.

Effects of multi-components released from S-PRG filler on the activities of human dental pulp-derived stem cells.

Numerous studies have shown that the sustained release of ions from dental restorative materials have acid buffering capacity, prevents tooth enamel demineralization, and inhibits bacterial adhesion. Herein, the release behavior and bioresponsiveness of ions released from surface pre-reacted glass-ionomer (S-PRG) fillers were investigated in different types of media based on human dental pulp-derived stem cell (hDPSC) responses. The hDPSCs were cultured for 1-7 days in S-PRG eluates diluted with varying amounts of cell culture media. S-PRG released several types of ions, such as F-, Sr2+, Na+, Al3+, BO33-, and SiO32-. The balance of eluted ions differed depending on the dilution and solvent, which in turn affected the cytotoxicity, cell morphology, cell proliferation, and alkane phosphatase activity of hDPSCs, among other properties. The results suggest that tailored S-PRG filler eluates could be designed and prepared for application in dental practice.

Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) vcells caused by serum deprivation and hypoxia promotes immune suppressive activity of mesenchymal stromal cells.

OBJECTIVES: We have previously reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, produces interleukin (IL)-1α, which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal 10T1/2 cells. This study assessed the conditions promoting the production of IL-1α in Sq-1979-1 cells, which could further enhance the immunosuppressive function of 10T1/2 cells, and evaluated its expression in OSCC tissues. METHODS: The expression of IL-1α was examined by RT-PCR, western blotting, and enzyme-linked immune sorbent assay (ELISA). The interferon (IFN)- γ-producing capability of anti-CD3 antibody-stimulated mouse spleen cells co-cultured with 10T1/2 cells and conditioned medium (CM) from Sq-1979-1 cells was examined by ELISA. The function of IL-1α was examined using an anti-IL1α antibody. Immunohistochemical analysis of the OSCC tissues was performed. RESULTS: The production of IL-1α from Sq-1979-1 cells was synergistically enhanced in lower serum (0.5% or 1.0% FBS) at the transcriptional level, and under hypoxia (1.0% oxygen) at the release level compared to that in the control medium supplemented with 10% FBS under normoxia. The IFN-γ-producing capability of stimulated spleen cells co-cultured with 10T1/2 cells was significantly reduced in the CMs prepared with the lower serum or under hypoxia. These functions of CMs were completely abolished by the anti-IL-1α antibody. The expression of IL-1α in OSCC tissues was prominent in the midst of a carcinomatous cellular lesion or a nearby necrotic lesion, where a supply deficiency could occur. CONCLUSION: s: IL-1α production by Sq-1979-1 cells was synergistically augmented under low serum and hypoxic conditions, which could promote the immunosuppressive activity of mesenchymal cells.

Evaluation of the mechanical properties and biocompatibility of gypsum-containing calcium silicate cements.

Mineral trioxide aggregate (MTA) cement is widely used in the field of endodontic treatment. We herein synthesized calcium silicates from calcium carbonate and silicon dioxide, with the aim of reducing the cost associated with the MTA. Additionally, we prepared gypsum-containing calcium silicate cement to reduce the setting time while enhancing the mechanical strength. We evaluated the physical properties of this cement and investigated the response of human dental pulp stem cells (hDPSCs) grown in culture media containing cement eluate. Our results revealed that calcium silicates could be easily synthesized in lab-scale. Furthermore, we demonstrate that gypsum addition helps shorten the setting time while increasing the compressive strength of dental cements. The synthesized gypsum-containing calcium silicate cement showed minimal cytotoxicity and did not inhibit the proliferation of hDPSCs. These results suggested that the newly developed calcium silicate material could be a promising pulp capping material.

Shikonin induces odontoblastic differentiation of dental pulp stem cells via AKT-mTOR signaling in the presence of CD44.

Purpose: In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation.Methods: We searched for a compound other than hyaluronan that induces odontoblastic differentiation of dental pulp stem cells and used western blotting to determine whether CD44 is involved in the induction of odontoblastic differentiation by the compound. We further validated the cell signaling details of the compound-induced expression of dentin sialophosphoprotein (DSPP), which is known as a marker of odontoblastic differentiation.Results: We investigated shikonin, which is one of the derivatives of naphthoquinone, the skeleton of vitamin K. Shikonin-induced expression of DSPP was inhibited by PI3K, AKT, and mTOR inhibitors. Additionally, shikonin-induced expression of DSPP was inhibited in dental pulp stem cells transfected with siRNA against CD44.Conclusions: Shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT-mTOR signaling pathway and CD44. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT-mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggests that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of the dental pulp.

Horseradish peroxidase interacts with the cell wall peptidoglycans on oral bacteria.

Salivary peroxidase and myeloperoxidase are known to display antibacterial activity against oral microbes, and previous indications have pointed to the possibility that horseradish peroxidase (HRP) adsorbs onto the membrane of the major oral streptococci, Streptococcus mutans and Streptococcus sanguinis (S. sanguinis). However, the mechanism of interaction between HRP and the bacterial cell wall component is unclear. Dental plaques containing salivary glycoproteins and extracellular microbial products are visualized with 'dental plaque disclosing agent', and are controlled within dental therapy. However, current 'dental plaque disclosing agents' are difficult to evaluate with just dental plaques, since they stain and disclose not only dental plaques but also pellicle formed with salivary glycoproteins on a tooth surface. In this present study, we have demonstrated that HRP interacted with the cell wall component of the major gram-positive bacterial peptidoglycan, but not the major cell wall component of gram-negative bacteria lipopolysaccharide. Furthermore, we observed that the adsorbed HRP labeled with fluorescence was detected on the major oral gram-positive strains S. sanguinis and Streptococcus salivarius (S. salivarius), but not on a gram-negative strain, Escherichia coli (E. coli). Furthermore, we have demonstrated that the combination of HRP and chromogenic substrate clearly disclosed the dental plaques and the biofilm developed by S. sanguinis, S. salivarius and the major gram-postive bacteria Lactobacillus casei on tooth surfaces, and slightly disclosed the biofilm by E. coli. The combination of HRP and chromogenic substrate did not stain either the dental pellicle with the salivary glycoprotein mucin, or naked tooth surfaces. These results have suggested the possibility that the adsorption activity of HRP not only contributes to the evaluation of dental plaque, but that enzymatic activity of HRP may also contribute to improve dental hygiene.

Immunomodulatory aspects in the progression and treatment of oral malignancy.

Inflammation substantially affects the risk of oral malignancy. Pro-inflammatory cytokine, interferon (IFN)-γ, confers anti-tumor activity using several different mechanisms. Conversely, higher expression of interleukin (IL)-17 is associated with worse prognosis. Monocyte chemotactic protein (MCP)-1 correlates positively with poor long-term survival of head and neck squamous cell carcinoma (HNSCC) patients. IL-1α affects cancer associated fibroblasts and macrophages, and promote several malignant phenotypes including immune suppression. Some anti-inflammatory cytokines, including IL-10 and transforming growth factor (TGF)-ß, relate to pro-tumoral activities. Among immune checkpoint modulators, programmed death (PD-)1 and PD-ligand (L)1 facilitate oral squamous cell carcinoma (OSCC) cell evasion from immune surveillance, and the expression status of these has a prognostic value. OSCCs contain tumor associated macrophages (TAMs) as major stromal cells of their tumor microenvironment. Among the two distinctive states, M2 macrophages support tumor invasion, metastasis and immune suppression. Crosstalk between TAMs and OSCC or cancer-associated fibroblasts (CAF) plays an important role in the progression of OSCC. Clinical trials with blocking antibodies against IL-1α or melanoma-associated antigens have been reported as therapeutic approaches against OSCCs. The most promising approach activating antitumor immunity is the blockade of PD-1/PD-L1 axis. Manipulating the polarization of pro-tumorigenic macrophages has been reported as a novel therapeutic approach.

Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) induces neuronal cell death by activating p53 pathway following traumatic brain injury.

Overactivation of N-methyl-d-aspartate glutamate receptors (NMDARs) after traumatic brain injury (TBI) contributes to excitotoxic cell death. The hyperactivation of NMDARs results in toxic levels of intracellular Ca2+ and in the activation of p53-mediated apoptosis pathway. Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) was identified as an epileptogenic gene of unknown function in our laboratory. In this study, we investigated the expression and cellular localization of NCDAP1 in rat models of fluid percussion-induced TBI. NCDAP1 expression increased in the ipsilateral cortex and hippocampus adjacent to the lesion of the TBI rats compared with that in the sham-operated controls. In addition, NCDAP1 was co-expressed with neuronal marker (NeuN), and the results of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining suggest that NCDAP1 is involved in neuronal apoptosis that occurs after brain injury. In addition, the expression levels of p53, Bax, and active caspase-3 correlated with those of NCDAP1. To further investigate the function of NCDAP1, primary cultured neurons were employed to establish an apoptosis model. The expression of NCDAP1 was induced by NMDA-induced Ca2+ influx, and the knockdown of NCDAP1 by siRNA decreased apoptosis caused by treatment with NMDA. Silencing of NCDAP1 also reduced p53 expression, whereas the over-expression of NCDAP1 induced cell death and up-regulated the expression of p53. The inhibition of p53 with pifithrin alpha or siRNA counteracted the effects of NCDAP1. Based on our data, we suggest that NCDAP1 plays an important role in p53-mediated neuronal apoptosis following TBI.

The luminance ratio of autofluorescence in a xenograft mouse model is stable through tumor growth stages.

The aim of this research was to investigate the value of autofluorescence imaging of oral cancer across different stages of tumor growth, to assist in detecting tumors. A xenograft mouse model was created with human oral squamous cell carcinoma cell line HSC-3 being subcutaneously inoculated into nude mice. Tumor imaging was performed with an autofluorescence imaging method (Illumiscan®) using the luminance ratio, which was defined as the luminance of the tumor site over the luminance of normal skin tissue normalized to a value of 1.0. This luminance ratio was continuously observed postinoculation. Tumor and normal skin tissues were harvested, and differences in the concentrations of flavin adenine dinucleotide and nicotinamide adenine dinucleotide were examined. The luminance ratio of the tumor sites was 0.85 ± 0.05, and there was no significant change in the ratio over time, even if the tumor proliferated and expanded. Furthermore, flavin adenine dinucleotide and nicotinamide adenine dinucleotide were significantly lower in tumor tissue than in normal skin tissue. A luminance ratio under 0.90 indicates a high possibility of tumor, irrespective of the tumor growth stage. However, this cutoff value was determined using a xenograft mouse model and therefore requires further validation before being used in clinical diagnosis.

Suppression effect on IFN-γ of adipose tissue-derived mesenchymal stem cells isolated from ß2-microglobulin-deficient mice.

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is a possible treatment for graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation and other inflammatory conditions. To address the mechanism of immunosuppression by MSCs, in particular those derived from adipose tissue (AMSCs), AMSCs were isolated from three different mouse strains, and the suppressive capacity of the AMSCs thus obtained to suppress interferon (IFN)-γ generation in mixed lymphocyte reaction cultures serving as an in vitro model of GVHD were assessed. It was revealed that the AMSCs had a potent capacity to suppress IFN-γ production regardless of their strain of origin and that such suppression was not associated with production of interleukin-10. In addition, the results demonstrated that ß2-microglobulin (ß2m)-deficient AMSCs from ß2m-/- mice were also potent suppressor cells, verifying the fact that the mechanism underlying the suppression by AMSCs is independent of major histocompatibility complex (MHC) class I expression or MHC compatibility. As AMSCs appear to have immunosuppressive properties, AMSCs may be a useful source of biological suppressor cells for the control of GVHD in humans.

Perspectives of Immune Suppression in the Tumor Microenvironment Promoting Oral Malignancy.

INTRODUCTION: In order to survive, cancers control immune systems and evade immune detection using mediators consisting of immune checkpoint molecules and cellular systems associated with immune suppression. METHODOLOGY: During the development of cancer and chronic infections, the immune checkpoints and cellular components including regulatory T cells, myeloid derived suppressor cells and cancer associated fibroblasts are often enhanced as a mechanism of immune subversion and have therefore become very important therapeutic targets. CONCLUSION: In this review, we will discuss the complexity of immune-suppressive mechanisms in the tumor milieu of cancers, including oral malignancy.

Gene expression analyses associated with malignant phenotypes of metastatic sub-clones derived from a mouse oral squamous cell carcinoma Sq-1979 cell line.

To elucidate the genetic events that occur during the development of OSCC, the present study established a model of oral malignancy using a mouse oral squamous cell carcinoma (OSCC) Sq-1979 cell line. Sq-1979 cells were implanted into syngeneic C3H mice. Subsequently, 233 cells and metastatic sub-clones (L cells) from primary OSCC, as well as the metastasized lymph node tissues of Sq-1979-implanted mice were established. Compared with parental Sq-1979 and 233 cells, the majority of L cells exhibited a higher proliferation rate and transplantability, and conferred a lower survival rate on the implanted mice. To investigate the genetic background of L cells, preferentially expressed genes in L cells were identified by cDNA microarray and reverse transcription-polymerase chain reaction analyses. The expression of FYN-binding protein (Fyb), solute carrier family 16 member 13 (Slc16a13), keratin 7, transmembrane portion 173 and Slc44a3 mRNAs was significantly elevated in L cells compared with that in Sq1979 and 233 cells. The mRNA expression was also evaluated in human OSCC and leukoplakia (LP) tissues. Among the 5 aforementioned mRNAs, the expression of FYB and SLC16A13 was significantly higher in OSCC than in LP tissues. Furthermore, the expression of SLC16A13 mRNA was significantly elevated in highly invasive OSCCs, which were classified as grades 3 and 4 by the Yamamoto-Kohama (YK) classification of invasion, compared with those in lower grades (YK-1 and -2). The model proposed in the present study could thus describe essential marker genes for the diagnosis of oral malignancies.

Tumor growth limited to subcutaneous site vs tumor growth in pulmonary site exhibit differential effects on systemic immunities.

To evaluate systemic immunity associated with tumor growth limited to a subcutaneous site versus growth proceeding at multiple tumor sites, we established syngeneic mouse subcutaneous and pulmonary tumor models by local subcutaneous and intravenous injection of colon carcinoma CT26 cells. We found that splenic myeloid-derived suppressor cell (MDSC) levels were significantly increased in the subcutaneous tumor model but not in the pulmonary tumor model. Furthermore, both CD4+ and CD8+ T cells as well as CD4+ Foxp3+ T cells were significantly decreased in the subcutaneous tumor model and were largely unchanged in the pulmonary tumor model. In addition, the subcutaneous model, but not the pulmonary model, displayed a Th1 polarization bias. This bias was characterized by decreased IL-4, IL-9, and IL-10 production, whereas the pulmonary model displayed increased production of IL-10. These results suggest that the mode of tumor development has differential effects on systemic immunity that may, in turn, influence approaches to treatment of cancer patients.

Metastasized murine oral squamous cell carcinoma cells induce intratumoral polymorphonuclear myeloid derived suppressor cells.

Myeloid derived suppressor cells (MDSCs) localize to hematopoietic organs and peripheral blood during inflammation or tumor tissues and lymph nodes in the presence of a tumor. However, whether there are differences in MDSCs found in the primary tumor and metastases is unknown. In the present study, we established a cell line of metastasized tumor cells to a lymph node, L5-11, which were derived from the Sq-1979 mouse buccal mucosa squamous cell carcinoma cell line. We then analyzed tumor immunogenicity, especially with regard to MDSCs, to clarify the differences between the primary tumor and metastases, using an isogenic heterotopic tumor transplantation model. Our data showed that the population of intratumoral MDSCs, especially polymorphonuclear MDSCs in the lymph node metastasis model were significantly increased compared with syngeneic grafts from the primary cell line Sq-1979 after 21 days. Furthermore, we identified that the lymph node metastasis cell line had increased expression of genes that promote the expansion of MDSCs, tumor growth and metastasis. Hence, these data suggest that tumor immunosuppression can occur via activation of MDSCs. However, further examination is required to clarify whether all or a subset of these factors from the lymph node metastasis tumor cells are required to induce intratumoral MDSCs.

Promotion of Nickel (Ni) Allergy by Anamnestic Sensitization with a Bacterial Component, Lipopolysaccharide (LPS), in Mice.

BACKGROUND/OBJECTIVE: Lipopolysaccharides (LPS) promote allergic responses to nickel (Ni) both in the sensitization and elicitation steps. In this study, we examine the effect of pre-sensitization to LPS on the occurrence of Ni allergy using a mouse model. METHOD: A 100 mg of LPS was injected into C57BL/6J mice intraperitoneally (ip). Three weeks later, the mice were subsequently injected with 0.3 µ moles of nickel dichloride (NiCl2) and 100 µg of CpG-DNA, which acted as an adjuvant. The mice were repeatedly immunized with the 0.3 µg of nickel sulfate (NiSO4), along with 300 µl of the adjuvant, Inject Alum (Pierce, USA). Then we examined the producing capabilities of T helper type 1 (Th1) and 2 (Th2) cytokines (interferon-gamma- (IFN)-γ and interleukin (IL)-10, respectively) from anti CD3 antibody-stimulated spleen cells. RESULTS: Pre-treatment with LPS, followed by repeated challenges with Ni2+ and adjuvants significantly enhanced the IFN-γ-producing capability of spleen cells (n=5, p<0.01); however, that could not enhance the capability of spleen cells by a single challenge with Ni2+ and adjuvants (n=5). In contrast, without LPS treatment, single or even repeated challenges by Ni2+ could not enhance the IFN-γ-producing capability. On the other hand, the IL-10-producing capability of spleen cells was not enhanced even by LPS and repeated challenges with Ni2+ and adjuvants. CONCLUSION: The solitary pre-sensitization to LPS is essential for the onset of Ni allergy by shifting the Th1/Th2 immune balance toward a Th1 dominant.