Treatment with lysophosphatidic acid improves glomerulonephritis through the suppression of macrophage activation in a murine model of systemic lupus erythematosus.

OBJECTIVES: Several therapeutic agents have been developed and used for the clinical treatment of systemic lupus erythematosus (SLE). In cases where SLE is accompanied by severe organ failures, such as neuropsychiatric lupus erythematosus (NPSLE) and acute onset of lupus nephritis, the use of potent immunosuppressive drugs, such as cyclophosphamide, is necessary. However, potent immunosuppressive drugs are known to increase infection risks. Thus, the development of therapeutic agents with novel mechanisms is urgently required. Previously, we reported that treatment with lysophosphatidic acid (LPA) prevents depression-like behaviours by suppressing microglial activation in MRL/lpr mice. In this study, we examined whether the treatment with LPA improves glomerulonephritis by affecting systemic immunity in MRL/lpr mice. METHODS: Eighteen-week-old MRL/lpr mice were treated with a vehicle or LPA for 3 weeks. After treatment, the glomerular inflammation and damage parameters were compared between the 2 groups. Moreover, we examined the effects of LPA on immune cells by flow cytometry using isolated splenocytes. RESULTS: LPA treatment in MRL/lpr mice significantly reduced the daily urinary albumin content and suppressed the CD68-positive cells and Periodic acid-Schiff (PAS)-positive areas in the glomeruli. The treatment also suppressed plasma anti-dsDNA antibodies and inflammatory cytokines in MRL/lpr mice. Although LPA did not significantly affect the total number of splenocytes, the treatment significantly reduced CD11b+Ly6G-Ly6C- cells (mature macrophages), as well as CD11b+Ly6G-Ly6C-CD68+ cells (activated mature macrophages). CONCLUSIONS: These results suggest that LPA may improve glomerulonephritis by suppressing macrophage activation in MRL/lpr mice.

Cytokine Expression and Macrophage Localization in Xenograft and Allograft Tumor Models Stimulated with Lipopolysaccharide.

T cell-deficient mice such as nude mice are often used to generate tumor xenograft for the development of anticancer agents. However, the functionality of the other immune cells including macrophages, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) in the xenograft are largely unknown. Macrophages and dendritic cells (DCs) acquire functionally distinct properties in response to various environmental stimuli; the interaction of these cells with MDSCs in tumor microenvironments regulates cancer progression. Nude mice are less likely to reject human cancer cells because of major histocompatibility complex (MHC) mismatches. The tumor microenvironment in a xenograft, comprising human and mouse cells, exhibits more complex bidirectional signaling and function than that of allograft. Here, we evaluated the differences of myeloid cells between them. Plasma interferon-γ and interleukin-18 concentrations in the xenograft tumor model after lipopolysaccharide (LPS) administration were significantly higher than those in the allograft tumor model. MHC class I, II, and CD80 expression levels were increased in CD11b⁺ and MDSC populations after LPS administration in the spleen of a xenograft tumor model but not in that of an allograft tumor model. Additionally, the number of CD80- and mannose receptor C type 1 (MRC1)-expressing cells was decreased upon LPS administration in the tumor of the xenograft tumor. These results suggest that functions of macrophages and DCs are sustained in the xenograft, whereas their functions in response to LPS were suppressed in the allograft. The findings will encourage the consideration of the effects of myeloid cells in the xenograft for drug development.

The long elusive IgM Fc receptor, FcµR.

IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both pre-immune "natural" and antigen-induced "immune" IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcµR). We have recently identified a bona fide FcµR in both humans and mice. In this article we briefly review what we have learned so far about FcµR.

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.

Evaluation of skin sensitization based on interleukin-2 promoter activation in Jurkat cells.

Skin sensitization is an allergic reaction caused by certain chemical substances, and is an important factor to be taken into consideration when evaluating the safety of numerous types of products. Although animal testing has long been used to evaluate skin sensitization, the recent trend to regulate such testing has led to the development and use of alternative methods. Skin sensitization reactions are summarized in the form of an adverse outcome pathway consisting of four key events (KE), including covalent binding to skin proteins (KE1), keratinocyte activation (KE2), and dendritic cell activation (KE3). Equivalent alternative methods have been developed for KE1 to KE3, but no valid alternative has yet been developed for the evaluation of KE4 and T-cell activation. Current alternative methods rely on data from KE1 to KE3 to predict the effect of chemicals on skin sensitization. The addition of KE4 data is expected to improve the accuracy and reproducibility of such predictions. The aim of this study was to establish an assay to evaluate KE4 T-cell activation to supplement data on skin sensitization related to KE4. To evaluate T-cell activation, the Jurkat T-cell line stably expressing luciferase downstream of the pro-inflammatory cytokine interleukin-2 promoter was used. After exposure to known skin sensitizing agents and control substances, luciferase activity measurements revealed that this assay was valid for evaluating skin sensitization. However, two skin sensitizers known to have immunosuppressive effects on T-cells reacted negatively in this assay. The results revealed that this assay simultaneously allows for monitoring of the skin sensitization and immuno-suppressiveness of chemical substances and supplements KE4 T-cell activation data, and may thus contribute to reducing the use of animal experiments.

Relationship between Psychological Stress Determined by Voice Analysis and Periodontal Status: A Cohort Study.

In modern society, evaluation and management of psychological stress may be important for the prevention of periodontal disease. The purpose of this study was to examine the relationship between psychological stress (vitality and mental activity) evaluated by Mind Monitoring System (MIMOSYS) and periodontal status. Forty students of Okayama University underwent the oral examination and self-reported questionnaire on the first day (baseline) and the 14th day (follow-up). Voice recording was performed every day with the MIMOSYS app during the whole study period. The participants completed the Patient Health Questionnaire (PHQ)-9 and Beck Depression Inventory (BDI) at baseline and at follow-up. Spearman's rank correlation coefficient was used to determine the significance of correlations among variables. The PHQ-9 and BDI scores were negatively correlated with vitality in the morning. Change in vitality in the morning was significantly correlated with changes in periodontal inflammation. Mental activity was significantly correlated with change in mean probing pocket depth. This result shows that measurement of psychological stress using a voice-based tool to assess mental health may contribute to the early detection of periodontal disease.

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.

Detection of Major Depressive Disorder Based on a Combination of Voice Features: An Exploratory Approach.

In general, it is common knowledge that people's feelings are reflected in their voice and facial expressions. This research work focuses on developing techniques for diagnosing depression based on acoustic properties of the voice. In this study, we developed a composite index of vocal acoustic properties that can be used for depression detection. Voice recordings were collected from patients undergoing outpatient treatment for major depressive disorder at a hospital or clinic following a physician's diagnosis. Numerous features were extracted from the collected audio data using openSMILE software. Furthermore, qualitatively similar features were combined using principal component analysis. The resulting components were incorporated as parameters in a logistic regression based classifier, which achieved a diagnostic accuracy of ~90% on the training set and ~80% on the test set. Lastly, the proposed metric could serve as a new measure for evaluation of major depressive disorder.

Simultaneous detection of T lymphocyte-related antigens (CD4/CD8, CD57, TCRß) with nuclei by fluorescence-based immunohistochemistry in paraffin-embedded human lymph node, liver cancer and stomach cancer.

OBJECTIVE: To develop a method for immunohistochemical staining of three different T lymphocyte antigens (CD4 or CD8, CD57 and TCRß) on the same tissue section and to determine whether tissues have been infiltrated with T lymphocytes expressing these markers. STUDY DESIGN: Commercially available antibodies were tested for immunohistochemical usefulness in a dye-based conventional single-immunostaining method after antigen retrieval on paraffin-embedded human lymph nodes. We searched for the combination of antibodies that could detect T lymphocyte antigens on the same section without any cross-reactivity and that have fluorescent signals robust enough to overcome paraffin autofluorescence. RESULTS: Application of the antigen retrieval technique and the Sudan black B quenching technique enabled staining of paraffin-embedded tissue sections with fluorescent-labeled secondary antibodies. The combination of primary and secondary antibodies that could simultaneously detect the T lymphocyte antigens CD4 or CD8, CD57 and TCRß in histochemical analysis of a paraffin-embedded human lymph node section was established, and was successfully applied to a human tissue section infiltrated with T lymphocytes that express these markers. CONCLUSION: The antibodies listed here would be helpful for histopathologists who wish to investigate T lymphocytes in the paraffin-embedded sections that have accumulated in pathology labs throughout the world.

Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

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.

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.

Highly reliable, targeted photothermal cancer therapy combined with thermal dosimetry using a near-infrared absorbent.

Photothermal therapy (PTT) using a photo-absorbent in the near-infrared (NIR) region is an effective methodology for local cancer treatment. Before PTT using a NIR absorbent is executed, the operator generally determines the two parameters of fluence rate and irradiation time. However, even if the irradiation parameters are unchanged, the therapeutic effect of PTT is often different for individual tumors. Hence, we examined the therapeutic effect of PTT using a NIR absorbent (ICG lactosome) while changing two parameters (fluence rate and irradiation time) in various combinations. As a result, there was no robust correlation between those parameters and the therapeutic effect. Compared to those parameters, we found that a more reliable determinant was maintenance of the tumor temperature above 43 °C during NIR irradiation. To reconfirm the significance of the determinant, we developed a new system that can regulate the temperature at the NIR irradiation site at a constant level. By using the new system, we verified the treatment outcomes for tumors in which the NIR absorbent had accumulated. All of the tumors that had been kept at 43 °C during NIR irradiation were cured, while none of the tumors that had been kept at a temperature below 41 °C were cured. In conclusion, PTT using a NIR absorbent with thermal dosimetry is a highly reliable treatment for cancer.

PIRs mediate innate myeloid cell memory to nonself MHC molecules.

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

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.

Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

The level of serum CCL5, a C-C chemokine, is reportedly correlated with tumor progression in several cancers. We herein investigated the mechanisms by which CCL5 might contribute to tumor progression in gastric cancer. Serum CCL5 levels significantly correlated with tumor progression and prognosis in patients with gastric cancer. Immunohistochemistry showed that tumor-infiltrating lymphocytes expressed CCL5, while the tumor cells expressed the CCL5 receptors. Fluorescent double staining showed that tumor-infiltrating CD4+ cells rather than CD8+ cells preferentially expressed CCL5. Using gastric cancer cell lines (MKN45, KATO III), we examined CCL5 production by coculturing whole peripheral blood mononuclear cells (PBMCs), CD4+ cells, or CD8+ cells, with tumor cells. CD4+ cells cocultured with tumor cells remarkably enhanced CCL5 production in a direct cell-cell contact manner over other cocultured PBMCs, including CD8+ cells. Gastric cancer cell lines expressed CCL5 receptors and augmented their proliferation in response to CCL5 stimulation. Furthermore, we examined the effect of CCL5-treated cancer cells on the cocultured PBMCs, focusing on the CD4+/CD8+ proportion and apoptosis. Coculture of CCL5-treated gastric cancer cells with PBMCs resulted in a significant decrease in the proportion of CD8+ cells but not CD4+ cells, suggesting Fas-FasL-mediated apoptosis in CD8+ cells. In immunodeficient mice coinjected with KATO III and PBMCs, neutralization of CCL5 significantly suppressed tumor progression, resulting in a favorable outcome. In conclusion, gastric cancer cells might thus induce CD4+ T cells to secrete CCL5 and exploit it for their progression, as well as to aid in the prevention of CD8+ T cell-involved tumor elimination.

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.

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.

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.