Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis.

The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.

Human breast carcinoma cDNA encoding a galactoside-binding lectin homologous to mouse Mac-2 antigen.

A galactoside-binding lectin (Mr 29,000) has previously been identified in rat, mouse and human tissues. It is an abundant cell-surface component of inflammatory macrophages and their major non-integrin laminin-binding protein. It has also been found in the nucleus of other cell types. Here, we report the cloning and sequencing of a cDNA encoding the human galactoside-binding lectin from a breast carcinoma. The clone encodes a protein of 250 amino acids (aa) that is over 80% identical to its mouse and rat counterparts. The aa sequence has an N-terminal and a C-terminal, 'carbohydrate-binding', domain. The N-terminal domain consists of two parts. The first 41 aa are homologous to a transcription factor, i.e., the serum response factor. The adjacent part (aa 42-106) contains an unusual repeating element, that occurs seven times in human protein compared to nine times in rat and mouse. The C-terminal 'carbohydrate-binding' domain (aa 115-250) shows homology to L-14, another galactoside-binding lectin.

Gene expression, signal transduction and tissue-specific biomineralization during mammalian tooth development.

Tooth development provides a paradigm for intrinsic molecular controls for cell- and extracellular matrix (ECM)-mediated biomineralization. The intent of this review is to evaluate the sequential timing and positional information prerequisite for tissue-specific biomineralization. Recent investigations suggest that 1,25-dihydroxyvitamin D3 functions to up-regulate VDR (vitamin D receptor) that in turn could induce structural gene products, including calcium-binding proteins and several ECM proteins (e.g., enamelins, amelogenins, dentine sialoglycoproteins (DSP) and dentine phosphoproteins (DPP)), resulting in dentine and enamel formation. Inhibition of regulatory gene products and/or their receptors likely results in hypoplastic and/or hypomineralized ECM as a direct consequence of down-regulated (1) transcription and/or translation of structural and regulatory genes, (2) posttranslational modifications, (3) and/or decreased calcium transport to the forming dentine and enamel matrices. Advances in serumless in vitro culture methodology; computer-assisted access to nucleic acid sequences for probes to define when, where, and how many specific regulatory and structural gene products are expressed; antisense oligodeoxynucleotides to inhibit specific translation; and microtechniques to analyze biomineralization all provide additional avenues to investigate tissue-specific biomineralization.

Purification and characterization of tissue-type plasminogen activator in maxillary mucosa with chronic inflammation.

Tissue-type plasminogen activator (TPA) was purified from maxillary mucosa with chronic inflammation and compared with urokinase. Purification procedure consisted of the extraction from delipidated mucosa with 0.3M potassium acetate buffer (pH 4.2), 66% saturation of ammonium sulfate, zinc chelate-Sepharose, concanavalin A-Sepharose and Sephadex G-100 gel filtration chromatographies. The molecular weight of the TPA was approximately 58,000 +/- 3,000. Its activity was enhanced in the presence of fibrin and was quenched by placental urokinase inhibitor, but not quenched by anti-urokinase antibody. The TPA made no precipitin line against anti-urokinase antibody, while urokinase did. All these findings indicate that the TPA in maxillary mucosa with chronic inflammation is immunologically dissimilar to urokinase and in its affinity for fibrin.

Reconstruction of pleomorphic adenoma of the salivary glands in three-dimensional collagen gel matrix culture.

The morphogenesis of salivary gland pleomorphic adenoma was examined in vitro using three-dimensional (3-D) collagen gel culture. Pleomorphic adenoma cells were isolated from three parotid gland tumours and cultured as monolayers, after which they were subcultured in floating-collagen gel sandwiches. Cells cultured in both conditions were immunohistochemically characterized and compared using antibodies against various proteins representative of each histological component of salivary glands. Monolayers had myoepithelial characteristics, being positive for vimentin and alpha-smooth muscle actin. In collagen gels, however, the cells assembled in epithelial nests, showing an architecture similar to that of pleomorphic adenoma. The nests were composed of duct-lining epithelial cells that were positive for epithelial markers, surrounded by myoepithelial cells. Collagen gel culture induces multidirectional differentiation of adenoma cells, suggesting that pleomorphic adenomas originate from stem or reserve cells.

Involvement of tenascin-C in proliferation and migration of laryngeal carcinoma cells.

Tenascin-C (TN-C) is an extracellular matrix glycoprotein upregulated in various pathological processes. In this study, we investigated its distribution in dysplasia and carcinoma of the human larynx using immunohistochemistry and in situ hybridization (ISH) techniques. In all cancer tissues, TN-C immunostaining was markedly increased in the stroma, especially around the cancer cell nests. In addition, cytoplasmic staining of cancer cells was also observed in 62.5% of the invasive cases, the cells being distributed in the periphery of the nests adjacent to the stroma. TN-C mRNA signals in cancer cells were detected in all six cases examined by ISH. Furthermore, in vitro evaluation of the roles of TN-C demonstrated an increase in the proliferating cell fraction in a dose-dependent manner. In a wound closure assay, the addition of TN-C promoted migration. We conclude that TN-C secreted by cancer cells may be involved in their proliferation and migration in an autocrine fashion.

Tropoelastin expression by periodontal fibroblasts.

Elastic system fibers are load-bearing proteins found in periodontal tissue. There are three types--oxytalan, elaunin, and elastic fibers--which differ in their relative microfibril and elastin contents. Oxytalan fibers are known to be distributed in the periodontal ligaments and gingiva, whereas elaunin and elastic fibers are present only in the gingiva. We examined gene expression and accumulation of tropoelastin in the cell-matrix layers of human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF) in vitro. HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 8 wks. Northern blotting and RT-PCR analyses showed that only HGF expressed mRNA encoding tropoelastin. Western blotting analysis demonstrated 77-kDa protropoelastin and 68-kDa tropoelastin only in the cell-matrix layer of HGF cultured for 8 wks. These results suggest that the different tropoelastin expression patterns reflect the difference between HGF and HPLF phenotypes.

In vitro effects of calcitonin and/or parathyroid hormone on odontogenesis of mouse embryonic molars.

Mandibular first molars of mouse embryos were cultivated for examination of the effects of calcitonin (CT) and/or parathyroid hormone (PTH) on the odontogenesis of the molars, and for determination of whether and how CT, which is a PTH antagonist, has an influence on the effect of PTH on odontogenesis. On the second day, the inner enamel epithelium in the control group had already differentiated into pre-ameloblasts. Typical odontoblasts had secreted a layer of predentin. On the fourth day of culture, the pre-ameloblasts achieved terminal differentiation into secretory ameloblasts, and enamel and dentin had already been deposited. PTH (1 unit/mL) inhibited the odontogenesis of the cultured molars during the designated culture periods (two and four days), while CT (0.5 unit/mL) stimulated odontogenesis. On the second day, the development of the molars in the CT + PTH group showed an intermediate stage between the control and PTH-treated explants, but on day 4 it corresponded to that of the controls. Moreover, when the molars exposed to PTH for two days were untreated and treated with CT for an additional two days, the former produced a small quantity of enamel matrix, while the latter formed a large amount of the matrix. These histological findings were also supported by a morphometric analysis of the enamel matrix in the cultured molars. The present results suggest that CT stimulates, but PTH suppresses, the odontogenesis of the mouse embryonic molars, and that CT is an antagonist to the inhibitory effect of PTH on odontogenesis.

Tumor necrosis factor gene polymorphism in chronic sinusitis.

OBJECTIVES: It is likely that genetic factors play a role in the etiology of chronic sinusitis, and airway inflammation is an important pathological feature in chronic sinusitis. We hypothesized that individuals with greater inflammatory responses may be more likely to acquire the disease. Polymorphisms of the tumor necrosis factor (TNF) genes have been described, and certain inflammatory diseases are reportedly associated with certain alleles of TNF genes. The purpose of this study is to examine whether there is an association between some alleles of TNF genes and chronic sinusitis. STUDY DESIGN: Thirty-eight Japanese patients with intractable chronic sinusitis were selected on the basis of the following criteria: 1) persistent mucous or mucopurulent nasal discharge and/or postnasal dripping for longer than 3 years and 2) opacification in bilateral maxillary sinuses and ethmoid cells on plain radiographic films. METHODS: Both tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) gene polymorphisms were analyzed by polymerase chain reaction (PCR) with restriction fragment length polymorphisms in these patients and 35 healthy control subjects. RESULTS: A significantly higher frequency (P < .05) of TNFB*2 allele of TNF-beta gene polymorphism was observed in patients with chronic sinusitis (74%) compared with control subjects (56%). There was no association between alleles of TNF-alpha and chronic sinusitis. CONCLUSION: We concluded that TNF-beta gene polymorphism may form a component of the genetic predisposition to chronic sinusitis in Japanese patients.

Quantitative study of nasal secretory cells in normal subjects and patients with chronic sinusitis.

The contribution of nasal secretory cells to mucus hypersecretion in chronic sinusitis was investigated. The mucosae of the inferior turbinate were obtained from 18 normal control subjects and 65 patients with chronic sinusitis. Histochemical quantitation showed that there was no significant difference in the number of goblet cells between normal controls and chronic sinusitis. On the other hand, the number of submucosal acinar cells in chronic sinusitis was significantly higher than that in normal controls (P < 0.01). The area occupied by the acini in lamina propria was also increased in chronic sinusitis (P < 0.01). There was no significant difference in the distribution of the intra-acinar glycoproteins between normal control subjects and patients with chronic sinusitis. Results suggest that hyperplasia and hypertrophy of nasal acinar cells may have an important role in mucus hypersecretion in chronic sinusitis.

Quantitative cytology of nasal secretions and scrapings in Japanese cedar pollinosis patients.

To explore the dynamics of the cellular response to natural allergen exposure, the authors of this study performed quantitative cytology in both the nasal secretions and nasal scrapings of 16 normal control subjects and 23 Japanese cedar pollinosis patients before and during the pollen season. In nasal scrapings, the number of metachromatic cells increased significantly during the pollen season, although there was no significant difference between the normal subjects and the preseason pollinosis patients. The cell differential count in the patients with pollinosis showed that lymphocytes were predominant in scrapings; however, the majority of inflammatory cells in the nasal secretions were neutrophils before the season and eosinophils during the season. The study findings suggest that the appearance of metachromatic cells in nasal scrapings is specific to the pollen exposure and that the cytology in nasal secretions is significantly different from that in nasal scrapings.

Analysis of HLA antigens in Japanese patients with chronic sinusitis.

OBJECTIVES: Genetic factors likely play a role in the etiology of chronic sinusitis and this disease is often associated with diffuse panbronchiolitis, which is strongly associated with HLA B54 antigen. The purpose of this study is to examine whether genetic factors are involved in the pathogenesis of chronic sinusitis. STUDY DESIGN: Eighty-two Japanese patients with intractable chronic sinusitis were selected on the basis of the following criteria: 1) persistent mucous or mucopurulent nasal discharge and/or postnasal dripping for longer than 3 years and 2) opacification in bilateral maxillary sinuses and ethmoid cells in plain X-ray films. METHODS: Both class I and class II HLA antigens were analyzed by conventional microcytotoxicity assays in these patients and 176 healthy control subjects. RESULTS: In class I antigens, B54 antigen significantly increased in the patient group (antigen frequency = 29.3%, relative risk = 3.23, corrected P value = .037) compared with normal control group (antigen frequency = 11.4%). For class II antigens, no antigens were significantly increased. CONCLUSION: These data indicate that certain genetic factors play a role in the etiology of chronic sinusitis.

Quantitative cytology of nasal secretions with perennial allergic rhinitis in children: comparison of noninfected and infected conditions.

This study was performed to quantify the number of inflammatory cells in nasal secretions from pediatric patients with perennial allergic rhinitis under noninfected and infected conditions. Nasal secretions were obtained from seven children under both noninfected and infected conditions with perennial allergic rhinitis to house dust mites, and secondary quantitative cytology was performed on the secretions. The number of neutrophils under infected condition was significantly higher than that under noninfected condition (P < .05), whereas the number of eosinophils in infected condition was significantly lower than that in noninfected condition (P < .05). The ratio of eosinophil count to neutrophil count was more than 0.1 in noninfected condition. The ratio was significantly decreased in infected condition (P < .02). These results suggest that the distribution of inflammatory cells in the nasal mucus of children with allergic rhinitis would be modified under infected condition.

Quantitative analysis of mucin and lectin in maxillary sinus fluids in patients with acute and chronic sinusitis.

OBJECTIVES: Sinusitis is characterized by quantitative and qualitative changes in mucus biosynthesis that contribute to sinus disease. In general, patients with acute sinusitis complain of purulent rhinorrhea, whereas those with chronic sinusitis complain of mucoid or mucopurulent rhinorrhea Locally produced mucin largely contributes to the high viscoelasticity of mucus in sinusitis. In this study, the authors attempt to quantify the concentrations of mucin and lectin in the maxillary sinus fluids from these patients. STUDY DESIGN: To assess the concentrations of mucin and lectin in aspirates of 11 acutely and 11 chronically inflamed maxillary sinuses, the concentration of mucin was measured by enzyme-linked immunosorbent assay (ELISA) and that of lectin by sandwiched enzyme-linked lectin assay (ELLA). RESULTS: The concentrations of mucin and lectin that were measured using monoclonal antibodies (HCS 4, HCS 14, and HCS 18) and Ulex europaeus agglutinin-1 (UEA-1) in chronic sinusitis were higher than those in acute sinusitis. The concentration of total protein in chronic sinusitis was lower than that in acute sinusitis. Each mucin and lectin versus total protein ratio in chronic sinusitis was higher than that in acute sinusitis. CONCLUSIONS: These data suggest that hypersecretion in chronic sinusitis may result from locally increased mucin production. Comprehension of this mechanism may be a strategy to prevent the viscous cycle of paranasal sinus fluids in chronic sinusitis.

Quantitative cytology of nasal secretions under various conditions.

Quantitative cytology was performed in nasal secretions of normal control (NC), seasonal allergic rhinitis in season (SAR), perennial allergic rhinitis (PAR), chronic sinusitis with mucoid secretion (MS), and chronic sinusitis with mucopurulent secretion (MPS). The majority of inflammatory cells were neutrophils in NC, MS, and MPS; the majority were eosinophils in SAR and PAR. The concomitant appearance of inflammatory cells in nasal secretions was found, i.e., there were significant correlations between neutrophil and eosinophil counts in MPS, and between eosinophil and basophil counts in SAR. The eosinophil/neutrophil ratio was more than 0.1 in SAR and PAR, but the ratio was less than 0.1 in all NC, all MPS, and in 93% of MS; this indicates that 0.1 in eosinophil/neutrophil ratio is the critical value between allergic and nonallergic nasal diseases.

Nasal mucociliary transport of chronic sinusitis in children.

Nasal mucociliary function is one of the most important and indispensable mechanisms of the respiratory tract, providing protection against the atmospheric environment. We previously found mucociliary dysfunction in the noses of adult patients suffering from chronic sinusitis. In this study, using the saccharin method, we determined nasal mucociliary function in normal children and in children with chronic sinusitis. The mean (+/- SD) value of saccharin transit time in the nose was 28.2 +/- 19.9 minutes in patients with chronic sinusitis, this being significantly slower than that in the control group of children. The incidence of abnormally slow nasal mucociliary transport time (> 30 minutes) in patients was significantly higher than in controls of the same age. Mucociliary dysfunction may initiate a vicious cycle of self-mediated inflammation and may be important in recovery from chronic respiratory inflammation.

Effects of functional endoscopic sinus surgery on maxillary sinus mucosa.

OBJECTIVE: To compare the preoperative and postoperative changes on ciliary surface of maxillary sinus mucosa in patients treated with functional endoscopic sinus surgery. DESIGN: The maxillary mucosa of both the superolateral wall and the ostium were sampled during the operation and 6 to 12 months (mean duration, 7.6 months) after the operation. Ciliary surface was determined using scanning electron microscopy in combination with an image analyzer and was expressed in terms of ciliary area, which is the percentage of mucosal surface occupied by cilia. SETTING: The samples were taken at a hospital-based clinic. An electron microscopic study was performed at Mie University School of Medicine, Mie, Japan. PATIENTS: Sixteen patients (20 maxillary sinuses) undergoing functional endoscopic sinus surgery for treatment of chronic sinusitis. RESULTS: The mean (+/-SD) ciliary area before the surgery was 60.7%+/-28.8% and 39.9%+/-21.5% in the superolateral wall of the maxillary sinus and the ostium of the maxillary sinus, respectively. The ciliary area of the superolateral wall was significantly higher than that of the ostium (P<.001). The mean (+/-SD) postoperative ciliary area value was 74.3%+/-22.6% in the superolateral wall and 51.3%+/-16.1% in the ostium. These postoperative values were significantly higher than the preoperative values (P<.001). CONCLUSIONS: This study showed that the maxillary sinus mucosa in chronic sinusitis is capable of regeneration, and the damaged ciliated epithelium could return toward normal with the improvement of ventilation and drainage of the maxillary sinus following functional endoscopic sinus surgery.

Aneurysm of the extracranial carotid artery.

Aneurysms of the extracranial carotid artery are rare vascular lesions. These aneurysms usually present to the otolaryngologist or vascular surgeon as a cervical or parapharyngeal pulsatile mass. Rupture and hemorrhage are unusual complications. Central nervous system symptoms secondary to embolism or thrombosis, however, are relatively common. Because of these serious complications, surgical resection of the aneurysm with restoration of arterial continuity is the treatment of choice. We describe herein three cases of the extracranial carotid aneurysm treated at the Mie University Hospital, Tsu, Japan, in the past two years. Surgical treatments include end-to-end anastomosis, saphenous vein autograft replacement, and direct closure utilizing an internal shunt, respectively, after resection of the aneurysm. In all patients, the postoperative course was uneventful, and angiography six weeks after the operation demonstrated good blood flow through the repaired artery.