The Soluble Factor from Oral Cancer Cell Lines Inhibits Interferon-γ Production by OK-432 via the CD40/CD40 Ligand Pathway.

(1) Background: OK-432 is a penicillin-killed, lyophilized formulation of a low-toxicity strain (Su) of Streptococcus pyogenes (Group A). It is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously showed that (i) OK-432 treatment induces a high amount of IFN-? production from peripheral blood mononuclear cells (PBMCs), and (ii) conditioned medium (CM) from oral cancer cells suppresses both the IFN-? production and cytotoxic activity of PBMCs driven by OK-432. The aim of this study was to determine the inhibitory mechanism of OK-432-induced IFN-? production from PBMCs by CM. (2) Methods: We performed cDNA microarray analysis, quantitative RT-PCR, and ELISA to reveal the inhibitory mechanism of CM. (3) Results: We found that CD40 plays a key role in IFN-? production via IL-12 production. Although OK-432 treatment upregulated the expression levels of the IL-12p40, p35, and CD40 genes, CM from oral cancer cells downregulate these genes. The amount of IFN-? production by OK-432 treatment was decreased by an anti-CD40 neutralizing antibody. (4) Conclusions: Our study suggests that uncertain soluble factor(s) produced from oral cancer cells may inhibit IFN-? production from PBMCs via suppressing the CD40/CD40L-IL-12 axis.

Physical and Histological Comparison of Hydroxyapatite, Carbonate Apatite, and ß-Tricalcium Phosphate Bone Substitutes.

Three commercially available artificial bone substitutes with different compositions, hydroxyapatite (HAp; Neobone®), carbonate apatite (CO3Ap; Cytrans®), and ß-tricalcium phosphate (ß-TCP; Cerasorb®), were compared with respect to their physical properties and tissue response to bone, using hybrid dogs. Both Neobone® (HAp) and Cerasorb® (ß-TCP) were porous, whereas Cytrans® (CO3Ap) was dense. Crystallite size and specific surface area (SSA) of Neobone® (HAp), Cytrans® (CO3Ap), and Cerasorb® (ß-TCP) were 75.4 ± 0.9 nm, 30.8 ± 0.8 nm, and 78.5 ± 7.5 nm, and 0.06 m²/g, 18.2 m²/g, and 1.0 m²/g, respectively. These values are consistent with the fact that both Neobone® (HAp) and Cerasorb® (ß-TCP) are sintered ceramics, whereas Cytrans® (CO3Ap) is fabricated in aqueous solution. Dissolution in pH 5.3 solution mimicking Howship's lacunae was fastest in CO3Ap (Cytrans®), whereas dissolution in pH 7.3 physiological solution was fastest in ß-TCP (Cerasorb®). These results indicated that CO3Ap is stable under physiological conditions and is resorbed at Howship's lacunae. Histological evaluation using hybrid dog mandible bone defect model revealed that new bone was formed from existing bone to the center of the bone defect when reconstructed with CO3Ap (Cytrans®) at week 4. The amount of bone increased at week 12, and resorption of the CO3Ap (Cytrans®) was confirmed. ß-TCP (Cerasorb®) showed limited bone formation at week 4. However, a larger amount of bone was observed at week 12. Among these three bone substitutes, CO3Ap (Cytrans®) demonstrated the highest level of new bone formation. These results indicate the possibility that bone substitutes with compositions similar to that of bone may have properties similar to those of bone.

Expression of CD44, CD44v9, ABCG2, CD24, Bmi-1 and ALDH1 in stage I and II oral squamous cell carcinoma and their association with clinicopathological factors.

Cancer stem cells (CSCs) exhibit self-replication, self-differentiation, drug resistance and immune evasion activities. In recent years CSCs have become increasingly important for the treatment of malignant tumors. CSCs express specific markers, including cluster of differentiation (CD)44, CD44 variant 9 (CD44v9), ATP-binding cassette sub-family G member 2 (ABCG2), CD24, B lymphoma Mo-MLV insertion region 1 homolog (BMI-1) and aldehyde dehydrogenase 1 (ALDH1). However, the prognostic value of their expression in patients with oral squamous cell carcinoma (OSCC) are not well known. The present study evaluated these markers in stage I and II patients with OSCC and examined the association between T classification, histological differentiation, classification of invasion mode, lymph node metastasis and disease-free survival rate. Tissue specimens were obtained from 70 patients with stage I or II OSCC following either surgery or biopsy. Immunohistochemistry was performed and positive staining was defiend as 10% positive cells. CD44 and CD44v9 expressions were strongly detected in all OSCC tissues compared with normal epithelial cells. A total of 22 (31.4%) cases expressed ABCG2 and there was a significant association between ABCG2 expression and invasion. A total of 41 cases (59.0%) expressed CD24 and there was a significant association between CD24 expression and invasion. A total of 33 cases (47.1%) expressed BMI-1 and there was a significant association between BMI-1 expression and the disease-free survival rate. A total of 18 cases (25.7%) expressed ALDH1. Although there was no association between ALDH1 expression and T classification, there were significant associations between ALDH1 expression and histological differentiation, invasion mode, metastasis and the disease-free survival rate. Multivariate analysis revealed that ALDH1 expression was the only prognostic factor for disease-free survival rate. The results of the present study suggest that the positivity of ALDH1 detected in patients with OSCC correlates with the number of cells undergoing epithelial mesenchymal transition and metastasis. These findings indicated that the expression of ALDH1 may be an effective prognostic marker indicating the survival of patients with stage I and II OSCC.

Fabrication and evaluation of carbonate apatite-coated calcium carbonate bone substitutes for bone tissue engineering.

Carbonate apatite-coated calcium carbonate (CO3 Ap/CaCO3 ) was fabricated through a dissolution-precipitation reaction using CaCO3 granules as a precursor to accelerate bone replacement based on superior osteoconductivity of the CO3 Ap shell, along with Ca2+ release from the CaCO3 core and quicker resorption of the CaCO3 core. In the present study, CaCO3 , 10% CO3 Ap/CaCO3 , 30% CO3 Ap/CaCO3 , and CO3 Ap granules were fabricated and examined histologically to evaluate their potential as bone substitutes. Larger contents of CaCO3 in the granules resulted in higher Ca2+ release and promoted cell proliferation of murine preosteoblasts at 6 days compared with CO3 Ap. Interestingly, in a rabbit femur defect model, 10% CO3 Ap/CaCO3 induced significantly higher new bone formation and higher material resorption compared with CO3 Ap at 8 weeks. Nevertheless, CO3 Ap showed a superior osteoconductive potential compared with 10% CO3 Ap/CaCO3 at 8 weeks. All tested granules were most likely resorbed by cell mediation including multinucleated giant cell functions. Therefore, we conclude that CO3 Ap/CaCO3 has a positive potential for bone tissue engineering based on well-controlled calcium release, bone formation, and material resorption.

Compositional and histological comparison of carbonate apatite fabricated by dissolution-precipitation reaction and Bio-Oss®.

Carbonate apatite (CO3Ap) is an inorganic component of bone. This study aimed to compare the composition and tissue response to of CO3Ap (CO3Ap-DP) fabricated by the dissolution-precipitation reaction using calcite as a precursor and Bio-Oss®, which is widely used in orthopedic and dental fields as a synthetic bone substitute. X-ray diffraction and Fourier transform infrared results showed that CO3Ap-DP and Bio-Oss® were both B-type carbonate apatite with low crystallinity. The average sizes of CO3Ap-DP and Bio-Oss® granules were 450 ± 58 and 667 ± 168µ m, respectively, and their carbonate contents were 12.1 ± 0.6 and 5.6 ± 0.1 wt%, respectively. CO3Ap-DP had a larger amount of CO3 than Bio-Oss® but higher crystallinity than Bio-Oss®. When a bone defect made at the femur of rabbits was reconstructed with CO3Ap-DP and Bio-Oss®, CO3Ap-DP granules were partially replaced with bone, whereas Bio-Oss® remained at 8 weeks after implantation. CO3Ap-DP granules elicited a significantly larger amount of new bone formation at the cortical bone portion than Bio-Oss® at 4 weeks after the implantation. The results obtained in the present study demonstrated that CO3Ap-DP and Bio-Oss® showed different behavior even though they were both classified as CO3Ap. The CO3 content in CO3Ap played a more important role than the crystallinity of CO3Ap for replacement to bone and high osteoconductivity.

Lingual tonsillolith: prevalence and imaging characteristics evaluated on 2244 pairs of panoramic radiographs and CT images.

OBJECTIVES: Lingual tonsilloliths are not as well-known to radiologists than palatine tonsilloliths, although they might be common in clinical practice. The aim of this investigation was to clarify the prevalence and imaging characteristics of lingual tonsilloliths using panoramic radiographs and CT images. METHODS: This study included 2244 patients without pathology at the base of tongue who had undergone panoramic radiography and CT of the maxillofacial region. The size, number and position of lingual tonsilloliths relative to the mandible and tongue were evaluated. RESULTS: Lingual tonsilloliths were observed in 33 (1.5%) and 108 (4.8%) of all patients on panoramic radiographs and CT images, respectively. The prevalence was higher in patients aged ≥40 years than in those aged < 40 years (χ2, p < 0.01). They appeared as small, round- or rod-shaped calcified bodies, and they always located closely anterior (1-17 mm) to the anterior border of oropharyngeal airway on panoramic radiographs. Lingual tonsilloliths were superimposed over the surrounding soft tissue inferior to the body of the mandible, posteroinferior to the angle of the mandible and posterior to the mandible in 16 (48.5%), 15 (45.5%) and 1 (3.0%) individual, respectively. A significant correlation was observed between the detectability on panoramic radiographs and size (Spearman's r = 0.961, p < 0.01) of tonsilloliths, as revealed by CT images. CONCLUSION: Lingual tonsilloliths commonly appear on CT. They also appear on panoramic radiography and may superimpose the surrounding soft tissue of the mandible. Although lingual tonsilloliths may resemble other pathological calcifications including submandibular sialoliths and lingual osseous cholistoma, they can be differentiated by carefully observing panoramic radiographs. When clinicians detect calcified bodies near the base of tongue, lingual tonsilloliths should be included in the differential diagnoses.

Prevalence and imaging characteristics of palatine tonsilloliths evaluated on 2244 pairs of panoramic radiographs and CT images.

OBJECTIVES: Palatine tonsilloliths incidentally detected on diagnostic imaging should be differentiated from pathologic calcifications to enable correct diagnosis and treatment. The aim of this study is to clarify the prevalence and imaging characteristics of palatine tonsilloliths on panoramic radiographs. MATERIALS AND METHODS: We retrospectively reviewed 2244 individuals who underwent pairs of consecutive panoramic radiography and computed tomography (CT) of the head and neck region. The imaging characteristics of palatine tonsilloliths on panoramic radiography were compared with the findings from CT, which was considered the gold standard. RESULTS: Tonsilloliths were detected in 300 (13.4 %) and 914 (40.7 %) of the 2244 individuals on panoramic radiographs and CT, respectively. On panoramic radiographs, tonsilloliths were superimposed over the ramus of the mandible at the level coincident with and inferior to the soft palate in 176 (7.8 %) and 90 (4.0 %) individuals, respectively. Tonsilloliths were also superimposed over the surrounding soft tissue inferior to the body of the mandible, postero-inferior to the angle of the mandible, and posterior to the ramus of the mandible in 33 (1.5 %), 26 (1.2 %), and 28 (1.3 %) individuals, respectively. A significant correlation was observed between the detectability on panoramic radiographs and the size (Spearman r = 1.000) and number (Spearman r = 0.991) of tonsilloliths, as revealed by CT images. CONCLUSIONS: The present results suggest that tonsilloliths are commonly detected on panoramic radiographs. Furthermore, they can be superimposed on both the mandible and the surrounding soft tissue. CLINICAL RELEVANCE: Clinicians should include tonsilloliths among the differential diagnoses when calcified bodies are detected on panoramic radiographs.

Allergic contact dermatitis caused by titanium screws and dental implants.

PATIENTS: Titanium has been considered to be a non-allergenic material. However, several studies have reported cases of metal allergy caused by titanium-containing materials. We describe a 69-year-old male for whom significant pathologic findings around dental implants had never been observed. He exhibited allergic symptoms (eczema) after orthopedic surgery. The titanium screws used in the orthopedic surgery that he underwent were removed 1 year later, but the eczema remained. After removal of dental implants, the eczema disappeared completely. DISCUSSION: Titanium is used not only for medical applications such as plastic surgery and/or dental implants, but also for paints, white pigments, photocatalysts, and various types of everyday goods. Most of the usage of titanium is in the form of titanium dioxide. This rapid expansion of titanium-containing products has increased percutaneous and permucosal exposure of titanium to the population. CONCLUSIONS: In general, allergic risk of titanium material is smaller than that of other metal materials. However, we suggest that pre-implant patients should be asked about a history of hypersensitivity reactions to metals, and patch testing should be recommended to patients who have experienced such reactions.

Fabrication and Physical Evaluation of Gelatin-Coated Carbonate Apatite Foam.

Carbonate apatite (CO3Ap) foam has gained much attention in recent years because of its ability to rapidly replace bone. However, its mechanical strength is extremely low for clinical use. In this study, to understand the potential of gelatin-reinforced CO3Ap foam for bone replacement, CO3Ap foam was reinforced with gelatin and the resulting physical characteristics were evaluated. The mechanical strength increased significantly with the gelatin reinforcement. The compressive strength of gelatin-free CO3Ap foam was 74 kPa whereas that of the gelatin-reinforced CO3Ap foam, fabricated using 30 mass % gelatin solution, was approximately 3 MPa. Heat treatment for crosslinking gelatin had little effect on the mechanical strength of the foam. The gelatin-reinforced foam did not maintain its shape when immersed in a saline solution as this promoted swelling of the gelatin; however, in the same conditions, the heat-treated gelatin-reinforced foam proved to be stable. It is concluded, therefore, that heat treatment is the key to the fabrication of stable gelatin-reinforced CO3Ap foam.

Effects of low crystalline carbonate apatite on proliferation and osteoblastic differentiation of human bone marrow cells.

Carbonated apatite (CO3Ap) is the inorganic component of bone. We have proposed a new method for the fabrication of CO3Ap blocks based on a dissolution-precipitation method using a synthetic precursor. The aim of this study is to examine the effects of low crystalline CO3Ap on initial cell attachment, proliferation and osteoblastic differentiation of human bone marrow cells (hBMCs) using sintered hydroxyapatite and tissue culture plates as controls. Initial cell attachment and proliferation were assessed with a MTT assay. Expression of osteoblastic markers was examined by reverse transcription-polymerase chain reaction. XRD and FT-IR results showed formation of B-type carbonate apatite with lower crystallinity. No difference was observed for initial cell attachment between HAp and CO3Ap discs. hBMSC attached more significantly on tissue culture plate than on HAp and CO3Ap discs. The number of cells on HAp was higher than that on CO3Ap until day 7, after which the number of cells was similar. hBMSC proliferated more significantly on tissue culture plate than on HAp and CO3Ap discs. In contrast, hBMCs incubated on CO3Ap demonstrated much higher expression of osteoblastic markers of differentiation, such as type I collagen, alkaline phosphatase, osteopontin and osteocalcin, than hBMCs on HAp. On the tissue culture plate, they were not any change throughout the culture period. These results demonstrated that low crystalline CO3Ap exhibit higher osteoinductivity than HAp.

Antitumour effect of valproic acid against salivary gland cancer in vitro and in vivo.

Salivary gland cancer (SGC) has a comparatively poor prognosis and is prone to frequent recurrence and metastases. Therefore, the development of more effective chemotherapy against SGC is desirable. The aim of the present study was to investigate the antitumour effects of valproic acid (VPA) against SGC in vitro and in vivo. Two human SGC cell lines (HSY and HSG cells) were used in the present study. The effects of VPA on the proliferation of SGC cells in vitro were assessed by MTT assay. Cancer cells treated with VPA were subjected to cell cycle analysis by flow cytometry. In addition, the expression levels of p21 and p27 were examined by real-time RT-PCR to identify the mechanisms of the antitumour effect of VPA on SGC. The effects of VPA on cancer growth in vivo were evaluated in a xenograft model. VPA inhibited the proliferation of SGC cells in a dose-dependent manner in vitro. Degenerated cancer cells were observed at high concentrations of VPA. In the cell cycle analysis, VPA induced cell-growth inhibition and G1 arrest of cell cycle progression in both cancer cell lines in a time- and dose-dependent manner. VPA markedly upregulated the mRNA expression levels of both p21 and p27 in both SGC cell lines in a time-dependent manner. In the xenograft model experiment, VPA treatment markedly inhibited the growth of salivary gland tumours when compared with the growth of the untreated controls. VPA may be a valuable drug in the development of better therapeutic regimens for SGC.

Effect of soluble factors derived from oral cancer cells on the production of interferon-γ from peripheral blood mononuclear cells following stimulation with OK-432.

The streptococcal antitumor agent OK-432 is commonly used as an immunopotentiator for immunotherapy in various types of malignant tumors including oral cancer. It has been demonstrated that OK-432 elicits an antitumor effect by stimulating immunocompetent cells, thereby inducing multiple cytokines including interferon (IFN)-γ, interleukin (IL)-2 and IL-12. Serum concentrations of IFN-γ in patients with oral cancer were examined 24 h after administration of OK-432. Serum concentrations of IFN-γ in patients with advanced cancer were significantly lower than those in patients with early cancer. These results suggested that some soluble factors produced by cancer cells may inhibit IFN-γ production with OK-432. Thus, in the present study, an in vitro simulation model was established for the immune status of patients with oral cancer by adding conditioned medium (CM) derived from oral cancer cell lines into a culture of peripheral blood mononuclear cells (PBMCs) derived from a healthy volunteer. We investigated whether soluble factors derived from oral cancer cells affected IFN-γ production from PBMCs following stimulation with OK-432. PBMCs stimulated with OK-432 produced a large amount of IFN-γ; however, both IFN-γ production and cytotoxic activity from PBMCs induced by OK-432 were inhibited by the addition of CM in a dose-dependent manner. In order to examine these inhibitory effects against IFN-γ production, the contribution of inhibitory cytokines such as IL-4, IL-6, IL-10, transforming growth factor-ß and vascular endothelial growth factor was investigated. However, neutralization of these inhibitory cytokines did not recover IFN-γ production inhibited by CM. These results indicated that unknown molecules may inhibit IFN-γ production from PBMCs following stimulation with OK-432.

Bortezomib-enhanced radiosensitization through the suppression of radiation-induced nuclear factor-κB activity in human oral cancer cells.

Oral cancer cells have a significantly augmented nuclear factor-κB (NF-κB) activity and the inhibition of this activity suppresses tumor growth. Bortezomib is a proteasome inhibitor and a drug used for molecular-targeted therapy (targets NF-κB). In this study, we investigated whether bortezomib would be effective as an inhibitor of proliferation and a radiosensitizer for the treatment of oral cancer. We demonstrate that bortezomib inhibits NF-κB activity and cell proliferation. The combined treatment with bortezomib and radiation (RT) suppressed NF-κB activity and cell growth in vitro and in vivo compared with RT treatment alone. To investigate the mechanisms by which bortezomib suppresses tumor growth, the expression of signaling molecules downstream of NF-κB were examined by ELISA. The combined treatment significantly inhibited the radiation-induced production of angiogenic factors and decreased the number of blood vessels in the tumor tissues. Although the expression of anti-apoptotic proteins was upregulated by RT, bortezomib downregulated the RT-induced expression of these proteins. Moreover, the expression of cleaved poly(ADP-ribose) polymerase in vitro and in vivo was enhanced by bortezomib, indicating that bortezomib inhibits tumor growth by inducing apoptosis. This study clearly demonstrates that bortezomib significantly inhibits tumor growth and that the combined treatment with bortezomib and RT results in a significant inhibition of tumor growth. The mechanisms underlying the inhibition of tumor growth by bortezomib include the suppression of angiogenesis and the induction of apoptosis. A novel molecular targeting therapy including bortezomib may be effective in the treatment of oral cancer by suppressing NF-κB activity.

Expression and function of CXCR4 in human salivary gland cancers.

Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs.

Antitumor efficacy of sequential treatment with docetaxel and 5-fluorouracil against human oral cancer cells.

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.

[Evaluation of combination chemotherapy with oral S-1 administration followed by docetaxel by superselective intra-arterial infusion for patients with oral squamous cell carcinomas].

The purpose of this study was to evaluate the effectiveness and adverse events of combination chemotherapy with oral S-1 administration following docetaxel (DOC) treatment by superselective intra-arterial infusion as neo-adjuvant chemotherapy (NAC) for patients with oral squamous cell carcinoma. Thirteen patients were enrolled in this study (9 men and 4 women, with a mean age of 61. 0 years). All patients were given S-1 65mg/m(2) per day for 14 days, and DOC 40-50mg/m(2) by intraarterial infusion was administered. The locoregional response evaluated 3 weeks after administration was 100%, including a 69. 2% complete response. According to Oboshi and Shimosato's classification, histological evaluation of surgical specimens revealed that 3 cases were Grade II a, 4 cases Grade II b, 1 case Grade IV a, and 4 cases Grade IV c. The severe side effects were neutropenia and cerebral infarction. The present study suggests that combination chemotherapy with S-1 and DOC by superselective intra-arterial infusion would be an effective and safe regimen in NAC for oral squamous cell carcinomas.

Migration-stimulating factor as a novel biomarker in salivary gland tumours.

BACKGROUND: The identification of novel stratification biomarkers would benefit the clinical management of patients with salivary gland tumours. Migration-stimulating factor (MSF) is a potent stimulator of cell invasion, matrix remodelling and angiogenesis. The aim of this study was to determine whether MSF was expressed in salivary gland tumours and its potential value as a diagnostic biomarker. METHODS: Paraffin-embedded archival specimens of small salivary gland tumours were stained with an MSF-specific antibody. The specimens included 27 malignant and seven benign tumours; histologically normal salivary gland adjacent to the tumour was present in 16 specimens. MSF expression was assessed by consensus of 2-4 independent observers according to various indices, including 'overall MSF grade', 'percentage of area stained' and 'intensity of the staining'. The motogenic effect of MSF on a salivary gland tumour cell line, HSG, was examined in the transmembrane assay. RESULTS: Overall MSF expression increased significantly in a step-wise fashion from normal salivary gland to benign and malignant tumours (P = 0.04-0.0001); with moderate/strong positive specimens representing 6%, 33% and 74% of the normal, benign and malignant specimens, respectively. MSF was heterogeneously expressed in both carcinoma and stromal cell compartments, its expression being higher in malignant than benign tumours regarding various MSF indices. In tissue culture studies, exogenous MSF stimulated the migration of HSG cells. CONCLUSIONS: These immunohistochemical and functional studies suggest that MSF expression is a potentially useful biomarker of salivary gland tumour progression.

Migration Stimulating Factor (MSF) promotes fibroblast migration by inhibiting AKT.

The protein kinase AKT is activated strongly by many motogenic growth factors, yet has recently been shown capable of inhibiting migration in several cell types. Here we report that treatment with Migration Stimulating Factor (MSF), a truncated form of fibronectin that promotes the migration of many cell types, inhibits AKT activity in human fibroblasts and endothelial cells. In fibroblasts, treatment with either MSF or the AKT inhibitor, Akti-1/2, stimulated migration into 3D collagen gels to a similar extent and the effects of Akti-1/2 on migration could be blocked by the expression of an inhibitor-resistant mutant, AKT1 W80A. These data indicate that MSF promotes fibroblast migration, at least in part, by inhibiting the activity of AKT.

Differential involvement of TGF-beta1 in mediating the motogenic effects of TSP-1 on endothelial cells, fibroblasts and oral tumour cells.

The role of TSP-1 in tumour growth and angiogenesis remains controversial, with both stimulatory and inhibitory roles proposed. The effects of TSP-1 on the migration of endothelial cells, fibroblast and oral tumour cell lines were examined using the transmembrane assay. TSP-1 induced a bi-phasic effect on human and bovine endothelial cells: stimulation at low concentrations (0.1-10 microg/ml) and inhibition at high concentrations (25-100 microg/ml). FGF-2-stimulated endothelial cell migration was either further stimulated or inhibited by TSP-1, following the same bi-phasic dose response as in the absence of FGF-2. In contrast, TSP-1 stimulated the migration of human fibroblast and oral tumour cells in a dose dependent manner; a plateau was reached with 5-25 microg/ml and no inhibitory effect was observed. These effects were partly neutralised by antibodies to alphavbeta3 integrin. TGF-beta1 (0.1-200 ng/ml tested) mimicked the effects of TSP-1 on cell migration. Function-neutralising antibodies to TGF-beta1 completely abolished both the stimulatory and inhibitory effects of TSP-1 on endothelial migration, but had no effect on TSP-1-stimulated migration of fibroblast and oral tumour cells. The effects of TGF-beta1 were not affected by antibodies to TSP-1. These results indicate that the effects of TSP-1 on endothelial cell migration are mediated by TGF-beta1, whereas the effects on fibroblast and tumour cell migration are TGF-beta1-independent.

Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation, by using a monoclonal antibody TS-2 that neutralizes the interferon-gamma-inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction.

We have previously isolated a lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal agent, by affinity chromatography on a CNBr-activated Sepharose 4B bound TS-2 monoclonal antibody (mAb) that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. In the current study, we compared the cytokine-inducing and anti-tumor activities of OK-PSA, a TS-2-binding fraction, with those of OK-PTF, a TS-2-unbinding fraction, in order to determine the efficacy of OK-PSA for clinical use in affinity chromatography using TS-2. In the in vitro experiments using human peripheral blood mononuclear cells (PBMCs), OK-PSA markedly induced Th1-type cytokines, while interleukin (IL)-6 and IL-10, Th2-type cytokines, were induced by OK-PTF. Th1-cytokine induction by OK-PTF was not dose-dependent and was suppressed when PBMCs were treated with a high concentration of OK-PTF. In a mouse model, Th1 cytokines were also induced by OK-PSA and Th2 cytokines were induced by OK-PTF. Th2 cytokine-inducing activity of OK-PTF was accelerated in tumor-bearing mice relative to that in healthy mice. Although the anti-tumor effect of OK-PTF was statistically significant, it was much weaker than that of OK-PSA. A significant difference between the anti-tumor effect of OK-PSA and that of OK-PTF was observed (P<0.05). Finally, OK-PSA elicited its cytokine-inducing effect via Toll-like receptor (TLR) 4, whereas OK-PTF-induced signaling was mediated by both TLR2 and TLR4. These findings strongly suggested that the affinity chromatography using TS-2 is a useful strategy to separate the effective component for cancer therapy (OK-PSA) from other components.