A genomic sequence of the type II-A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system in Mycoplasma salivarium strain ATCC 29803.

INTRODUCTION: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems are RNA-mediated adaptive immune systems that actagainst invading genetic elements such as phages or plasmids. CRISPR/Cas systems exist in nearly half of bacteria. Mycoplasma salivarium is a commensal species of the oropharynx. The American Type Culture Collection maintains five M. salivarium strains: ATCC 14277, 23064, 23557, 29803, and 33130. The genome sequence of ATCC 23064 revealed that it has an incomplete CRISPR/Cas system. However, the genome sequences of the remaining strains have not been analyzed. METHODS: We performed polymerase chain reaction-amplicon sequencing and de novo genome sequencing to evaluate the presence of the CRISPR/Cas system in four strains. RESULTS: Only ATCC 29803 possessed cas1, cas2, cas9, and csn2 genes, a CRISPR array, and tracrRNA. The sequences of most components were identical between the CRISPR/Cas systems of ATCC 29803 and ATCC 23064, whereas the spacer sequences and a region of the cas9 gene were different. Unlike the CRISPR/Cas system of ATCC 23064, the cas9 gene of ATCC 29803 was not disrupted by the presence of stop codons. CONCLUSION: ATCC 29803 possesses genomic components required to express the type II-A CRISPR/Cas system, which potentially functions as an RNA-guided endonuclease.

Ultrastructural Changes during the Life Cycle of Mycoplasma salivarium in Oral Biopsies from Patients with Oral Leukoplakia.

Bacteria in genus Mycoplasma spp. are the smallest and simplest form of freely replicating bacteria, with 16 species known to infect humans. In the mouth, M. salivarium is the most frequently identified species. Mycoplasma spp. are parasites with small genomes. Although most of the Mycoplasma spp. that infect humans remain attached to the host cell surface throughout their life cycle, we have previously reported the presence of Mycoplasma salivarium in the epithelial cells of oral leukoplakia and oral lichen planus. However, the mechanism underlying the pathogenicity of M. salivarium has remained unclear. Further studies are needed to identify the process of infection of human cells and the stages in the life cycle of M. salivarium. Electron microscopy (EM) is the method of choice for morphological investigation of Mycoplasma spp. in cells or tissues. This study was performed to clarify and detail the ultrastructure of M. salivarium in tissue biopsies of oral mucosal leukoplakia, using three EM methods: (1) a standard EM processing method; (2) an ultracryotomy and immunolabeling method; and (3) the LR White resin post-embedding and immunolabeling method. This study included five oral leukoplakia tissue samples showing hyperplasia and hyperkeratosis. Although there was some variation in ultrastructural appearances between the three EM methods used, there were four ultrastructural appearances that are believed to reflect the stages of the M. salivarium life cycle in the epithelial cells of the oral mucosa: (1) small, electron-dense cellular-like structures or elementary bodies of M. salivarium; (2) large structures of M. salivarium; (3) M. salivarium organisms in cell division; (4) the sequence of events in the life cycle of M. salivarium that includes: (a) elementary bodies of M. salivarium deep in the oral mucosal epithelium; (b) replication by binary fission and daughter cell division from the elementary bodies; (c) maturation or degeneration of M. salivarium in the epithelial cells mainly in the upper part of the epithelium; and (d) death of the organisms in the granular and/or keratinized layer. These ultrastructural images may provide a useful reference for the identification of M. salivarium in diagnostic cytology or biopsy material.

Immunohistochemical detection of Mycoplasma salivarium in oral lichen planus tissue.

BACKGROUND: Oral lichen planus (OLP) is a T-cell-mediated inflammatory disease; however, its exact etiology is unknown. Hyperkeratosis is often observed in OLP lesions. Previous studies have revealed the localization of Mycoplasma salivarium in the epithelial cells of oral leukoplakia with hyperkeratosis. Herein, we investigated the presence of M. salivarium in OLP tissue by immunohistochemistry to determine the causative factor of OLP. METHODS: Forty-one formalin-fixed, paraffin-embedded samples obtained from 31 patients with OLP were examined. Ten samples of normal-appearing oral mucosa were used as controls. Immunohistochemistry (IHC) was performed using anti-M. salivarium monoclonal antibodies. RESULTS AND CONCLUSIONS: Mycoplasma salivarium was detected in the epithelium and lymphocyte infiltrate area in 24 of 41 OLP samples (58.5%). The bacteria were intracellularly localized in epithelial cells, while it was unclear whether they were also localized in lymphocyte cells or in the extracellular spaces among the lymphocytes in the subepithelial lymphocyte infiltrate area. Little or no staining was observed in the epithelium in the normal-appearing mucosa samples. Sawtooth rete ridge formation was observed in 21 OLP samples (51.2%), and a significant positive correlation between sawtooth rete ridge formation and IHC positivity was demonstrated. However, the role of M. salivarium in the epithelium and lamina propria of OLP tissue remains unknown.

Transforming growth factor-ß1 induces invasion ability of HSC-4 human oral squamous cell carcinoma cells through the Slug/Wnt-5b/MMP-10 signalling axis.

Molecular mechanism underlying the invasion of oral cancer cells remains to be clarified. We previously demonstrated that transforming growth factor-ß1 (TGF-ß1) induces the expression of mesenchymal markers in human oral squamous cell carcinoma HSC-4 cells. Intriguingly, the expression of the epithelial-mesenchymal transition-related transcription factor Slug was also significantly upregulated upon TGF-ß1 stimulation. However, the mechanism by which Slug transduces the TGF-ß1-induced signal to enhance the invasiveness of HSC-4 cells is poorly understood. Proteomic analysis revealed that the expression of matrix metalloproteinase (MMP)-10 was upregulated in TGF-ß1-stimulated cells. Additionally, a Boyden chamber assay revealed that the TGF-ß1-induced increase in invasiveness of HSC-4 cells was significantly inhibited by MMP-10 small interfering RNA (siRNA). Intriguingly, Slug siRNA suppressed TGF-ß1-induced expression of MMP-10. These results suggest that TGF-ß1 induces invasion in HSC-4 cells through the upregulation of MMP-10 expression in a Slug-dependent manner. On the other hand, Slug siRNA suppressed TGF-ß1-induced Wnt-5b expression. Wnt-5b significantly induced MMP-10 expression, whereas Wnt-5b siRNA suppressed the TGF-ß1-induced increase in invasiveness, suggesting that TGF-ß1-induced expression of MMP-10 and the resulting upregulation of invasiveness are mediated by Wnt-5b. Overall, these results suggest that TGF-ß1 stimulates HSC-4 cell invasion through the Slug/Wnt-5b/MMP-10 signalling axis.

In situ immunohistochemical detection of intracellular Mycoplasma salivarium in the epithelial cells of oral leukoplakia.

BACKGROUND: Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue. OBJECTIVE: Our aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry. DESIGN: We produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry. RESULTS: We obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivarium DNA in the epithelial cells of leukoplakia. CONCLUSION: Intracellular M. salivarium was identified in the epithelial cells of leukoplakia.

Cytopathologic diagnosis on joint lavage fluid for patients with temporomandibular joint disorders.

Temporomandibular joint (TMJ) disorders (TMD) are usually diagnosed based on the patient's clinical findings and the results of image investigations; however, understanding of the inflammatory process in TMJ is difficult. In addition, many of the TMJ disease types share common principal symptoms. Therefore, TMJ diseases in the early stage can be misdiagnosed with TMD. It is hypothesized that cytopathologic examination of the joint lavage fluids is useful in interpreting the TMD-associated inflammatory process from a cellular aspect. The aim of this study was to assess the TMJ lavage fluid cytopathologically in TMD patients. Thirty-nine patients, clinically diagnosed as TMD, were included in the present study. Clinical symptoms of the patients were recorded. Forty-four samples of TMJ lavage fluid were collected and paraffin-embedded cell sections were made by cell block tissue array method. Cytologic conditions in upper articular cavity of TMJ were cytopathologically diagnosed and were compared with the clinical symptoms of each patient. Cell components were detected in 22 of the 44 analyzed joint lavage fluids. There was a correlation between cytopathologic findings and clinical symptoms. Variety of cytopathology and inflammatory conditions in patients with similar clinical symptoms were also found. The results suggested that cytopathologic examination of the joint lavage fluids from TMD patients is helpful for gaining an understanding of the inner local conditions of TMJ at the cellular level.

Congenital peripheral developing odontoma accompanied by congenital teratomatous fibroma in a 9-month-old boy: a case report.

Peripheral odontoma is rare, and only two cases of congenital peripheral odontoma have been reported. Congenital oral fibroma is also rare. We describe a unique case of congenital peripheral developing odontoma accompanied by congenital teratomatous fibroma in an infant. Both tumors were difficult to detect on radiography. Two small masses were seen in the median anterior portion of the palatal mucosa of a 9-month-old boy. The masses had been present since birth and were surgically removed at age 28 months, when one of the masses had grown to a diameter of 8 mm. Histopathologic examination showed a fibrous lesion and a tooth germ-like rounded lesion composed of dental papilla, enamel organ, dentin, and cementum. Although congenital odontoma is rare, it should be considered when selecting appropriate treatment, as early radiographic detection is difficult.

Expression of Wilms' tumor 1 (WT1) in oral squamous cell carcinoma.

BACKGROUND: The product of the Wilms' tumor gene, WT1 protein, is a tumor antigen for various kinds of cancer, and WT1 peptide-based cancer immunotherapy is widely anticipated as a new possibility for cancer treatment. The aim of this study was to investigate the expression of WT1 from quantitative and morphological perspectives in oral squamous cell carcinoma (OSCC), the most widespread malignant neoplasm of the oral cavity. METHODS: Six OSCC cell lines and tissue sections from 29 OSCC patients were analyzed. To detect WT1 expression, reverse transcription-polymerase chain reaction analysis (RT-PCR), real-time PCR, Western blots, and immunofluorescence flow cytometry for WT1 were performed on the cell lines, and immunohistochemistry and fluorescence in situ hybridization (FISH) were performed on the tissue sections. RESULTS: WT1 mRNA was found overexpressed in one of the six OSCC cell lines, with expression levels higher than that seen in human leukemia cell line (K562). Immunohistochemical analysis of tissue sections showed overexpression of WT1 protein in two patients, concentrated mainly in the cytoplasm of the outer one to three cell layers of the cancer nests. This was consistent with the expression of WT1 mRNA observed by FISH. Meanwhile, WT1 was not detected on normal oral epithelium. WT1 protein was detected on actively proliferating cancer nests and even on elongated epithelial ridge where new droplet-cancer-nests were being formed and starting infiltration toward subepithelial layer. CONCLUSIONS: The results suggest that WT1 plays an important role in the pathogenesis of some types of OSCC, particularly in proliferation of the cancer cells.

Combined intra-arterial infusion and systemic chemoradiotherapy for stage IV squamous cell carcinoma of the mandibular gingiva.

PURPOSE: The purpose of this study was to show the effectiveness of combining intra-arterial infusion and systemic chemotherapy with concurrent radiotherapy for treatment of stage IV mandibular gingival cancer. MATERIALS AND METHODS: A total of 23 patients with mandibular gingival cancer were treated with either docetaxel by intra-arterial infusion followed by systemic chemoradiotherapy with cisplatinum and 5-fluorouracil as a monthly regimen, or with docetaxel and cisplatinum by intra-arterial infusion followed by systemic chemoradiotherapy with 5-fluorouracil as a weekly or biweekly regimen. Tumor responses, locoregional control, overall survival, disease-specific survival, and adverse events were evaluated. RESULTS: Of the 23 patients enrolled in the study, 22 completed the treatment. With regard to clinical stages, 82 % were diagnosed as IVA and 18 % IVB. Complete and partial response was observed in 82 and 18 %, respectively. Five-year overall survival, disease-specific survival, and locoregional control were 51, 70, and 72 %, respectively. No statistically significant difference was seen between the monthly regimen and the weekly plus biweekly regimen, although the latter resulted in longer survival and 88 % control. CONCLUSION: Combined intra-arterial infusion and systemic chemoradiotherapy may be an effective treatment for patients with stage IV mandibular gingival cancer.

Fibroblast growth factor-1-induced ERK1/2 signaling reciprocally regulates proliferation and smooth muscle cell differentiation of ligament-derived endothelial progenitor cell-like cells.

The periodontal ligament (PDL) is a fibrous connective tissue located between the tooth root and the alveolar bone. We previously demonstrated that a single cell-derived culture of primarily cultured PDL fibroblasts has the potential to construct an endothelial cell (EC) marker-positive blood vessel-like structure, suggesting that the fibroblastic lineage cells in ligament tissue could act as the endothelial progenitor cells (EPCs), which regenerate to construct a vascular system around the damaged ligament tissue. Moreover, we showed that EPC-like fibroblasts expressed not only EC markers but also smooth muscle cell (SMC) markers. Generally, an interaction between ECs and SMCs regulates blood vessel development and remodeling, and is required for the formation of a mature and functional vascular network. However, the mechanism underlying the SMC differentiation of the ligament-derived EPC-like fibroblasts remains to be clarified. In this study, we showed that suppression of fibroblast growth factor 1 (FGF-1)-induced extracellular signal-regulated kinase 1/2 (ERK1/2) signaling with the MAPK/ERK kinase (MEK) inhibitor U0126 completely abolished the FGF-1-induced proliferation of the ligament-derived EPC-like fibroblasts. In addition, U0126 treatment of FGF-1-stimulated ligament-derived EPC-like fibroblasts significantly induced the SMC differentiation of the cells. Thus, FGF-1-induced ERK1/2 signaling not only promoted the proliferation of the ligament-derived EPC-like fibroblasts, but also suppressed the SMC differentiation of the cells, suggesting that FGF-1 controls the construction of a vascular network around the ligament tissue by regulating the proliferation and SMC differentiation of the EPC-like cells through ERK-mediated signaling.

Establishment of human dental epithelial cell lines expressing ameloblastin and enamelin by transfection of hTERT and cdk4 cDNAs.

BACKGROUND: An in vitro cell culture system of dental epithelium is useful for the investigations of cellular differentiation and function of ameloblast in amelogenesis and of regenerative therapy in human tooth. However, there have been no immortalized human dental epithelial ameloblastic-lineage cell lines, which proliferate indefinitely and additionally produce enamel matrix proteins. METHODS: We transfected two retroviral constructs of human telomerase reverse transcriptase (hTERT) cDNA and mouse cyclin-dependent kinase 4 (cdk4) cDNA into the primary ameloblastoma cells and isolated immortalized human dental epithelial cell lines of HAM1, HAM2 and HAM3. The three cell lines were examined by electron microscopy, assay of senescence-associated ß-galactosidase activity, mRNA expression and immuno-reactivity of dental epithelial marker cell molecules and enamel matrix proteins. RESULTS: They showed undifferentiated phenotypes in monolayer culture and did not have any ß-galactosidase activity. The transcripts of dental epithelial cell markers of Msx2, Jagged1, Notch1, Sp3, Sp6, keratin 14 and keratin 18 were confirmed. In addition, mRNA and protein expression of ameloblastin and enamelin were also detected in three cell lines. All cells in the three cell lines were keratin 14- and 18-positive and some elongated cells were Jagged1-positive. Msx2-positive nuclei were noted in only HAM2 cells. CONCLUSION: We established three cell lines by transfection of hTERT and cdk4 cDNAs, which were characterized as dental epithelial progenitor cells containing ameloblast-lineage cell phenotype.

Epidemiological study of malignant tumors in the oral and maxillofacial region: survey of member institutions of the Japanese Society of Oral and Maxillofacial Surgeons, 2002.

We studied 1809 patients with oral cancer who visited and were treated, in 2002, at the 148 institutions certified as training facilities by the Japanese Society of Oral and Maxillofacial Surgeons. Of these institutions, 39 are dental university hospitals, 44 are medical university hospitals, 64 are general hospitals, and for 1 institution, the classification was not known. The patients consisted of 1071 (59.2%) males and 738 (40.8%) females (male: female ratio, 1.45:1), who had a average age of 65.2 years. The tongue (40.2%) was the most common site affected, followed by the gingiva (32.7%), buccal mucosa (10.1%), and oral floor (9.0%). There were 6 cases of multiple intraoral cancers. On histopathological examinations, squamous cell carcinoma (88.7%) was the most common type found, followed by adenoid cystic carcinoma (2.1%), and mucoepidermoid carcinoma (1.7%). Cases classified as T2N0 were the most common (32.1%), followed by T1N0 (21.4%), T4N0 (8.0%), and T2N1 (7.6%). Distant metastasis occurred in 17 patients (1.0%). Nonepithelial tumors, among which malignant melanoma was the most common type, accounted for 1.8% of the tumors. The sizes of the nonepithelial malignant tumors ranged from 1.0 to 7.0 cm, with an average size of 3.7 cm.