Dynamic changes in cell-surface expression of mannose in the oral epithelium during the development of graft-versus-host disease of the oral mucosa in rats.

BACKGROUND: The role of cell-surface glycoconjugates in oral mucosal graft-versus-host disease (GVHD) is still unclear, even though molecular changes in the oral epithelium are essential for the pathogenesis of these lesions. In this study, we investigated changes in the binding of mannose (Man)-specific Lens culinaris lectin (LCA) in the oral mucosa of rats with GVHD. METHODS: Lewis rat spleen cells were injected into (Lewis x Brown Norway) F1 rats to induce systemic GVHD, including oral mucosal lesions. Tongue and spleen samples were evaluated using lectin histochemistry, immunohistochemistry, Western blotting, transwell migration assays and Stamper-Woodruff binding assays. RESULTS: Binding of Man-specific LCA expanded to the epithelial layers of the tongue in GVHD-rats. An expansion of LCA binding was related to the increased expression of mannosyltransferase in the oral mucosa. CD8+ cells, effector cells of oral mucosal GVHD, expressed mannose-binding protein (MBP) and migrated to the medium containing Man in the transwell migration assay. Adherence of CD8+ cells to the oral epithelium could be inhibited by pretreating CD8+ cells with MBP antibody and/or by pretreating sections with Man-specific LCA. CONCLUSIONS: Increased expression of Man on keratinocytes leads to the migration and/or adhesion of CD8+ cells in the surface epithelium, which is mediated in part by the MBP/Man-binding pathway during the development of oral mucosal GVHD.

In vitro and in vivo expression of aldehyde dehydrogenase 1 in oral squamous cell carcinoma.

Aldehyde dehydrogenase isoform 1 (ALDH1) is a useful marker of cancer-initiating cells (CICs) in various organs. In this study, we investigated whether alterations in ALDH1 immunostaining and enzymatic activity in tumor cell populations predicted clinicopathological factors of prognostic importance for cancer progression and contributed to the characteristics of CICs in cisplatin-treated oral squamous cell cancer (OSCC) cells. We evaluated the association between the proportion of ALDH1-positive tumor cells and the clinicopathological features in 90 patients with OSCC. We also examined ALDH1 enzymatic activity, ABCG2 expression, invasive capacity and the ability to self-renew in OSCC cells treated with or without cisplatin. The clinicopathological results showed that elevated ALDH1 expression correlated with local recurrence. In in vitro experiments, the percentage of cells exhibiting ALDH1 enzymatic activity significantly increased among cisplatin-surviving cells (CiSCs) according to flow cytometry. Furthermore, CiSCs demonstrated upregulated expression of ABCG2, their invasive capacity increased, and their ability to generate cancer spheres was enhanced. An increased population of cells exhibiting ALDH1 immunostaining is a predictive marker of local recurrence. ALDH1 expression and activity contributes to the characteristics of CICs in OSCC.

Lupus-like oral mucosal lesions in mercury-induced autoimmune response in Brown Norway rats.

BACKGROUND: Administration of mercury at nontoxic doses induces systemic autoimmune disease in Brown Norway (BN) rats. The pathogenesis of lupus-like oral mucosal lesion by mercury-induced autoimmunity is still unclear, even though the oral mucosa is observed to be commonly affected in mercury-treated BN rats. In this study, we investigated the immunopathology of lupus-like oral mucosal lesions in a model of mercury-induced systemic autoimmunity. METHODS: Brown Norway male rats were injected subcutaneously with either phosphate-buffered saline (control) or mercury at a dose of 1.0 mg per kilogram of body weight on days 0, 3, 5, and 7. Blood, kidney, and tongue samples were taken at various timepoints for evaluation by immunohistochemistry, RT-PCR, and lupus band test (LBT). RESULTS: Oral mucosal lesions were classified according to three consecutive temporal phases on the basis of infiltration of immunocompetent cells as follows: (phase I) infiltration of MHC class II+ dendritic cells (DC) and macrophages; (phase II) addition of ED1+ macrophage infiltrates; and (phase III) focal infiltration of pan T cells following increased infiltration of DC and macrophages. Dense infiltration of DC and macrophages was observed in the basement membrane (BM) zone of the oral epithelium. Tissue expression of IL-4 mRNA was detected in early lesions (phase I), suggesting that locally produced IL-4 may be responsible for Th2-mediated immune response. A linear and continuous smooth pattern of fluorescence was observed in the oral epithelial BM in addition to renal glomeruli, indicating immune complex deposits. CONCLUSIONS: Local autoimmune responses are involved in the pathogenesis of mercury-induced lupus-like lesions of the oral mucosa.

Quantitative evaluation of myofibroblast apoptosis during wound healing in rat palate after post-operative administration of basic fibroblast growth factor (bFGF).

OBJECTIVE: Excessive wound contraction apparently inhibits maxillary growth; thus, myofibroblast apoptosis needs to be accelerated in mucoperiosteal denudation after palatoplasty. The aim of this study was to evaluate myofibroblast apoptosis during wound healing in mucoperiosteal denudation of rat palates immediately after post-operative administration of basic fibroblast growth factor (bFGF). MATERIALS AND METHODS: A total of 100 male Wistar rats aged 20 days were divided into control, scar, sham and bFGF groups (n = 25 each). In the scar, sham and bFGF groups, mucoperiosteum was removed from the palate and fibrin glue was applied to the exposed bone surface immediately after surgery. In the bFGF group, 10 µL of 2 µg/µL bFGF solution was injected into the operated area beneath the fibrin glue. At 2, 5, 7, 14 and 28 days post-operatively, myofibroblast apoptosis during the wound healing process was investigated by double immunofluorescence staining. The apoptotic area of myofibroblasts was measured using image software. RESULTS: In the bFGF group, at 2 days, apoptosis of myofibroblasts in the lamina propria and submucosa was marked, as compared with the other three groups and apoptosis of myofibroblasts was scarcely seen at 5 days. At 5 and 7 days, the apoptotic area of myofibroblasts in the bFGF group was statistically significantly smaller when compared to the scar and sham groups. CONCLUSION: The results confirmed that bFGF injection immediately after surgery accelerated apoptosis of myofibroblasts in mucoperiosteal denudation of rats. This may reduce maxillary growth retardation due to excessive wound contraction.

Tumor-induced lymphangiogenesis in cervical lymph nodes in oral melanoma-bearing mice.

BACKGROUND: Metastasis via the lymphatic system is promoted by lymphangiogenesis. Alterations of the lymphatic channels during the progression of metastasis to regional lymph nodes (LNs) remain unexplored. To examine whether tumor-induced LN lymphangiogenesis controls metastasis to regional LNs, we investigated cervical LN metastasis in a mouse model of oral melanoma. METHODS: Injection of B16F10 melanoma cells into mouse tongues replicated spontaneous cervical LN metastasis. We performed histological, immunofluorescent, and histomorphometric analyses of tumor-reactive lymphadenopathy and lymphangiogenesis in tumor-associated LNs. We investigated the expression of vascular endothelial growth factor (VEGF)-C and its receptor, VEGF receptor-3 (VEGFR-3), in tumor cells and tissues, and LNs by reverse transcription polymerase chain reaction and immunofluorescence. RESULTS: Tumor-associated LNs comprised sentinel LNs (SLNs) before and after tumor cell invasion (tumor-bearing SLNs), and LNs adjacent or contralateral to tumor-bearing SLNs. Extensive lymphangiogenesis appeared in SLNs before evidence of metastasis. After metastasis was established in SLNs, both LNs adjacent and contralateral to tumor-bearing SLNs demonstrated lymphangiogenesis. Interaction between VEGF-C-positive melanoma cells and VEGFR-3-positive lymphatic vessels was evident in tumor-associated LNs. CONCLUSIONS: LN lymphangiogenesis contributes a progression of tumor metastasis from SLNs to other regional LNs.

Immunohistochemical and Immunocytochemical Localization of Amylase in Rat Parotid Glands and von Ebner's Glands by Ion Etching-Immunoscanning Electron Microscopy.

The distribution of amylase in rat parotid glands and von Ebner's glands was examined using ion etching-immunoscanning electron microscopy, which enables both light and electron microscopic observations of identical semi-thin resin sections immunolabeled with anti-α-amylase and immunogold in association with silver enhancement. At the light microscopic level, most acinar secretory granules (SG) and striated duct secretions of parotid glands were strongly stained dark brown. In von Ebner's glands, acinar SG and duct secretions were weakly to strongly stained light to dark brown. At the electron microscopic level, labeling was observed as bright gold-silver particles. The labeling intensity of acinar SG of parotid glands was higher than that of von Ebner's glands. In parotid glands, weak labeling of SG in transitional cells between acini and intercalated ducts, very weak labeling of SG in intercalated ducts, and strong labeling of striated duct secretions were observed. In von Ebner's glands, the secretions and some SG of interlobular ducts were strongly labeled compared to those of intralobular ducts and SG of acini. Less amylase was synthesized in von Ebner's acini compared to parotid acini, whereas von Ebner's ducts may secrete significantly more amylase to modify saliva than parotid ducts.

Induction of epithelial migration of lymphocytes by intercellular adhesion molecule-1 in a rat model of oral mucosal graft-versus-host disease.

To elucidate the involvement of intercellular adhesion molecule-1 (ICAM-1) in the migration of lymphocytes to the oral mucosal epithelium in a rat model of acute graft-versus-host disease (AGVHD), we investigated (1) ICAM-1 and major histocompatibility complex (MHC) class II expression by keratinocytes (KCs) and their role in the epithelial infiltration of CD8+ cells, (2) the tissue expression of interferon-γ (IFN-γ) mRNA and expression of IFN-γ receptor by KCs, and (3) the ability of KCs to direct CD8+ cells into the epithelial layers. We classified the oral mucosal lesions into three consecutive temporal phases on the basis of increased epithelial ICAM-1 expression: basal- (phase I), parabasal- (phase II), and pan-epithelial except for the cornified cell layer (phase III). Basal ICAM-1 expression by KCs preceded that of MHC class II molecules, infiltration of CD8+ cells and epithelial histological changes. Tissue expression of IFN-γ mRNA and expression of IFN-γ receptor on KCs evidenced by immunohistochemistry were detected in early lesions (phase I), indicating that locally produced IFN-γ induced ICAM-1 expression by KCs. CD8+ cells were bound to KCs in frozen sections of epithelial lesions, whereas no lymphocyte attachment was observed in normal KC. Adherence could be inhibited by pretreating CD8+ cells with lymphocyte function-associated antigen-1 (LFA-1) antibody and/or by pretreating sections with ICAM-1 antibody. Our data suggest that in the early phase of acute oral mucosal GVHD, the induction of ICAM-1 expression on KCs leads to the migration of CD8+ cells into the epithelium and that this is mediated in part by the ICAM-1/LFA-1 pathway.

Influence of carbon dioxide laser irradiation on the healing process of extraction sockets.

OBJECTIVE: To clarify the healing-promoting effects of carbon dioxide laser irradiation in high and low reactive-level laser therapies (HLLT and LLLT, respectively) on extraction sockets after tooth extraction. MATERIAL AND METHODS: Forty-two 5-week-old male Wistar rats were divided into laser irradiation and non-irradiation (control) groups and compared. The laser-irradiation group underwent HLLT immediately after tooth extraction and then LLLT 1 day post-extraction. Tissue was excised 6 h and 3, 7, or 21 days after extraction and histopathologically investigated. The alveolar crest height was measured osteomorphometrically 21 days post-extraction, and granulation tissue in the extraction socket surface layer was immunohistologically investigated using anti-α-smooth muscle actin (anti-α-SMA) antibody 3 and 7 days post-extraction. RESULTS: Many osteoclasts appeared and active bone resorption was noted in the irradiation group 3 days after extraction compared to the controls. On Day 7, new bone formation started around the extraction socket in the control group, but from the superficial to over the middle layer of the socket in the irradiation group. On Day 21, a concavity existed in the alveolar crest region in the controls, whereas this region was flat, with no concavity, in the irradiation group. On osteomorphometry, the alveolar crest height was significantly higher in the irradiation (0.7791 ± 0.0122) than the control (0.6516 ± 0.0181) group (P < 0.05). On immunostaining, many α-SMA-positive cells were noted in the control group, but very few in the irradiation group. CONCLUSION: Laser-irradiated extraction wound healing showed characteristics different from those of the normal healing process, suggesting a favorable healing-promoting effect.

Solitary neurofibroma of the gingiva with prominent differentiation of Meissner bodies: a case report.

BACKGROUND: Oral neurofibromas are peripheral nerve sheath tumors, similar to schwannomas. Histological variations in oral neurofibromas are relatively uncommon. CASE PRESENTATION: Here, we present a case of unique variation in the observed characteristics of a neurofibroma, with no relation to neurofibromatosis type-1 or von Recklinghausen disease of the skin. The neurofibroma was observed in the right mandibular gingiva of a 32-year-old Japanese woman. Histologically, it differed from conventional neurofibromas in that the tumor was composed of a mixture of fine fibrillary collagen in sheets and/or cords of neoplastic Schwann cells containing numerous clusters of Meissner bodies. Histologically, these bodies were in contact with neoplastic Schwann cells. The Meissner bodies were immunopositive for S-100 protein, neuron-specific enolase, and vimentin, but were negative for calretinin. CD34-positive spindle cells were observed around the Meissner bodies. No recurrence or signs of other tumors have been observed in the patient for 5 years after tumor resection. CONCLUSION: To the best of our knowledge, no formal descriptions of sporadic, solitary neurofibromas containing numerous Meissner bodies occurring in the oral cavity are available in literature. We believe that an uncommon proliferation of Meissner bodies, as seen in the present case, may result from aberrant differentiation of neoplastic Schwann cells.

Specific expression of salivary maxi-K channel variant is augmented in diabetic mice.

BACKGROUND AND OBJECTIVE: A genetically diabetic mouse strain (db/db) exhibits severe obesity and a syndrome resembling human non-insulin-dependent diabetes mellitus. Our histological study of submandibular glands revealed that the size and area of the granular convoluted tubules was substantially decreased in db/db mice. We hypothesized that this structural difference reflected a specific alteration in salivary duct function. METHODS: The saliva evoked by pilocarpine was used for the measurement of ion concentrations, and submandibular glands were dissected out for the immunohistochemistry and real-time PCR study. RESULTS: The K(+) concentration of the salivary secretion was higher in db/db than in control m+/m+ mice, while neither saliva volume nor the concentrations of Na(+) or Cl⁻ differed between these strains. In db/db mice (vs. m+/m+ mice): quantitative PCR analysis revealed an increased mRNA expression of large-conductance Ca²(+)-activated K(+) (maxi-K) channels, immunohistochemistry revealed an increase in the luminal surface expression of the maxi-K channel protein, and a particularly interesting finding was that there was a substantial increase in the salivary tissue-specific splice variant ParSlo. CONCLUSION: These results suggest that in db/db mice, the K(+) content of saliva may be elevated due to an expression of a maxi-K channel variant, which results from a modification of ductal structure. GENERAL SIGNIFICANCE: Our data may shed some light on the mechanism responsible for determining the dynamics of salivary K(+) concentration increased in diabetic patients.

A poly(gamma-glutamic acid)-amphiphile complex as a novel nanovehicle for drug delivery system.

Recently, many studies have focused on biomedical and pharmaceutical applications of self-assembled nanoparticles. In addition, several biodegradable nanoparticles have been reported to possess poor dispersion stability and poor size-controllability. However, these nanoparticles require complicated fabrication procedures using synthesis techniques. We developed an efficient method for producing nanoparticles derived from a biological origin of molecule poly(gamma-glutamic acid) (gamma-PGA), a cationic lipid, and doxorubicin (Dox). The complex had a size of 510 nm and was able to encapsulate over 90% of the added Dox. An in vivo assay of antitumor activity demonstrated that the complex had significant antitumor activity in sarcoma 180-bearing mice, and was effectively accumulated in solid tumors based on the EPR effect. The data suggested that this complex is a promising formulation of gamma-PGA for targeted delivery to solid tumors. gamma-PGA-12GP2 complexes may possess several unique advantages, including simplicity of nanoparticle preparation, high drug-carrying capacity, appropriate size to allow deeper penetration based on EPR effect into solid tumors, and lack of necessity to modify the chemical structure of the drugs. These data indicate that the gamma-PGA-12GP2 complexes are potentially useful in cancer chemotherapy.

Effects of bFGF on suppression of collagen type I accumulation and scar tissue formation during wound healing after mucoperiosteal denudation of rat palate.

OBJECTIVE: The purpose of this study was to evaluate the effect of basic fibroblast growth factor (bFGF) on collagen changes after mucoperiosteal denudation of rat palate. MATERIAL AND METHODS: A total of 36 male Wistar rats were divided into control, scar, sham, and bFGF groups. In the scar, sham, and bFGF groups, lateral palatal mucoperiosteum was excised to form scar tissue on the palate. In the bFGF group, bFGF solution was injected into the operated area 1 week postoperatively. At 6 weeks postoperatively, the distribution of collagen type I and the 3-dimensional structure of collagen fibers were investigated under immunofluorescent and scanning electron microscopy. RESULT: In the bFGF group, weakly immunostained submucosa was clearly distinguishable from the strongly immunostained cervical periodontal ligament and gingiva. Collagen fibers running from submucosal tissue into the surface of underlying palatal bone comprised loosely arranged collagen fibrils. Lumen structures in collagen fibers resembled those in the control group. CONCLUSION: Administration of bFGF for suppression of collagen type I generation could suppress scar tissue formation and reduce connective strength with adjacent teeth and palatal bone.

Effects of basic fibroblast growth factor administration on vascular changes in wound healing of rat palates.

OBJECTIVE: The purpose of this study was to investigate the vascular changes induced by mucoperiosteal denudation of rat palate and to elucidate the effects of basic fibroblast growth factor (bFGF) administration on the palatal vascular network in wound healing. METHODS: A total of 117 male Wistar rats were used for the study on their 20th postnatal day. The animals were divided into three groups: a scar formation group, a basic fibroblast growth factor group, and a control group. The scar formation and basic fibroblast growth factor groups had lateral mucoperiosteum excised from the palate. In the basic fibroblast growth factor group, a solution of basic fibroblast growth factor was injected into the operated area 1 week after excision. At 6, 8, and 10 weeks postoperatively, palatal vascular changes were investigated by immunohistochemical staining and corrosion cast techniques. RESULTS: Throughout the experimental period, there were significantly fewer vessels in the scar formation group than in the control and basic fibroblast growth factor groups. In the basic fibroblast growth factor group, the elongation of new vessels and capillary proliferation proceeded, and after 10 weeks a highly organized vascular network was established. The scar formation group showed few Volkmann's canals that were shrunken or closed, whereas the basic fibroblast growth factor group evidenced Volkmann's canals with arterioles or venules, as seen in the control. CONCLUSIONS: The results suggested that injection of basic fibroblast growth factor into palatal wounds improves the vascular supply to the operated mucosa and underlying bone during and after palatal wound healing, which may contribute to tissue remodeling of the palate during growth.

Drug binding and releasing characteristics of DNA/lipid/PLGA film.

We evaluated the blended compound of DNA/lipid complexes and PLGA (poly(D,L-lactide-co-glycolide)) as a carrier material for drug delivery system (DDS). Transparent, self-standing DNA/lipid/PLGA films were prepared by casting from an organic solvent such as DMSO/chloroform. Daunorubicin hydrochloride (DH) could intercalate and groove bind into DNA in the films, whereby the amount of DH bound to the films was controlled by the latter's immersion period in DH aqueous solution. DH was released from DH films after immersion in PBS solution, whereby release rate was dependent on the chemical structure of lipids. Released DH caused reduction of cell viability during the cell culture of L929 mouse fibroblasts. These results suggested that DNA/lipid/PLGA film was a promising useful material for DDS.

Preparation of carbon nanotube-alginate nanocomposite gel for tissue engineering.

A novel scaffold material based on an alginate hydrogel which contained carbon nanotubes (CNTs) was prepared, and its mechanical property and biocompatibility evaluated. Soluble CNTs were prepared with acid treatment and dispersed in sodium alginate solution as a cross-linker. After which, the mechanical property (elastic deformation), saline sorption, histological reaction, and cell viability of the resultant nanocomposite gel (CNT-Alg gel) were evaluated. The CNT-Alg gel showed faster gelling and higher mechanical strength than the conventional alginate gel. Saline sorption amount of freeze-dried CNT-Alg gel was equal to that of the alginate gel. In terms of histological evaluation and cell viability assay, CNT-Alg gel exhibited a mild inflammatory response and non-cytotoxicity. These results thus suggested that CNT-Alg gel could be useful as a scaffold material in tissue engineering with the sidewalls of CNTs acting as active sites for chemical functionalization.

Study on rat subcutaneous reaction to experimental polyurethane elastomers.

The purpose of the study was to investigate the biocompatibility of experimental elastomers, E580 and E590. The experimental elastomers and the control--a clinically used elastomer--were implanted into the subcutaneous tissue of rats. The tissue reactions were examined histologically on the 3rd, 7th, 14th, 28th, and 56th day after implantation. It was found that there were some irritant responses in the tissues adjacent to the implanted elastomers during the first week. However, the inflammatory tissue reaction subsided substantially from the second week onwards. The stable fibrous capsule surrounding the elastomer was formed after eight weeks. The tissue responses of the control, E580, and E590 were similar. The results suggested that the long-term tissue irritation of the experimental elastomers was so low such that they have the potential to be applied clinically.

Central acinic cell carcinoma of the mandible. Case report.

Central acinic cell carcinoma (of the mandible) is rare, and, to our knowledge, only seven cases of this disease have been reported in the literature. A case in a 67-year-old Japanese woman is presented. Clinical examination revealed a 10.0x6.0mm mass located on the buccal aspect of the gingiva of the second molar in the left mandible. Radiographic examination revealed a radiolucency from the second to the third molar of the left mandible. Computed tomography disclosed destruction of the lingual cortical bone of the third molar region. The preliminary diagnosis was of odontogenic tumour. The patient was admitted, and removal of the tumour and of the involved teeth were carried out. Histological examination disclosed the diagnosis of acinic cell carcinoma. Subsequently, the tumour area was widely excised from the second premolar region to the coronoid process, and radical neck dissection was performed. A lymph node metastasis was found in the submandibular region. No recurrence or metastasis was observed during the 34-month follow-up.