Ascorbic acid enhances radiation-induced apoptosis in an HL60 human leukemia cell line.

This study was conducted to examine the utility of the combined use of ascorbic acid (AsA) and radiation in clinical applications. We investigated cell survival, DNA fragmentation, and caspase activation after X-ray irradiation and AsA treatment of human leukemia HL60 cells. The number of living cells decreased after combined X-ray irradiation and AsA treatment (2 Gy + 5 mM) in comparison with that after X-ray irradiation (2 Gy) or AsA treatment (5 mM) alone. DNA fragmentation was more in the cells subjected to combined X-ray irradiation and AsA treatment than in those subjected to X-ray irradiation alone. Caspase-3, caspase-8, and caspase-9 were highly activated following combined X-ray irradiation and AsA treatment, but caspase-8 activity was not markedly increased after X-ray irradiation alone. Bax levels in the mitochondrial membrane fractions were increased after AsA treatment alone and after combined X-ray irradiation and AsA treatment. However, there was no apparent increase in the Bax levels after X-ray irradiation treatment alone. Thus, this study confirmed that supplementing X-ray irradiation with AsA treatment results in increased apoptosis in HL60 cells. With regard to the apoptosis-inducing factors, we hypothesized that Bax and caspase-8 were activated after combined X-ray irradiation and AsA treatment compared with either treatment alone.

Nicotine induces upregulated expression of beta defensin-2 via the p38MAPK pathway in the HaCaT human keratinocyte cell line.

Human beta-defensins (hBDs), a group of antimicrobial peptides, are involved in the protective barrier of the oral epithelium. Nicotine induces periodontal and oral epithelial diseases. The purpose of the present study was to investigate the effect of nicotine on the expression pattern of hBD-2 in keratinocytes. HaCaT cells, a keratinocyte cell line, were incubated with 8, 15, 30, or 80 µM nicotine for 24 h. Expression of hBD-2 was observed by RT-PCR, qRTPCR, and ELISA assay. The cells were treated with inhibitors for intracellular pathways (p38MAP kinase, NF-κB, JNK, MAPK-ERK) and with nicotinic acetylcholine receptor (nAChR) inhibitors in a series of experiments. Data were analyzed using Student's t test. qRT-PCR revealed that the expression level of hBD-2 mRNA was significantly higher at 30 and 80 µM nicotine than the control without nicotine (P < 0.05). The 80 µM cell extraction contained significantly higher hBD-2 peptide levels than the control (P < 0.05). The p38MAP kinase inhibitor abolished the upregulated expression of hBD-2 by nicotine. Both nAChR inhibitors also abolished the upregulation of hBD-2 by nicotine. The present study demonstrated that nicotine causes upregulated expression of hBD-2 via the p38MAP kinase pathway in keratinocytes.

Expression of claudin-4 and -7 in porcine gingival junctional epithelium.

Junctional epithelium, a nonkeratinized stratified epithelium, extends apically in apposition to the surface of the enamel to form a seal between the epithelium and the tooth. Desmosomes and gap junctions adhere to the junctional epithelium through cell-cell contact, but no evidence of tight junctions has been found. Recently, tight junction hallmark proteins and tight junction-related structures have been identified in stratified squamous epithelium. The present study examined whether tight junction proteins were expressed in the junctional epithelium. We used immunohistochemical techniques to observe expression of claudin-1, -4, -5, -7, and occludin in porcine gingival junctional epithelium. Claudin-4 exhibited immunoreactivity in the intercellular spaces of all layers of the oral epithelium and the junctional epithelium. Stronger expression was observed in junctional epithelial cells adjacent to the inner and outer basal laminae than in the inner cell layers. Immunohistochemical positivity for claudin-7 was clearly observed in the junctional epithelium, but only a faint positivity was observed in the basal layer of the oral epithelium. No immunohistochemical positivity for claudin-1, -5, or occludin was observed in the junctional epithelium. RT-PCR assay confirmed expression of porcine claudin-4 and -7 mRNAs in the junctional epithelium. These findings indicate that claudin-4 and -7 may play a role in the junctional epithelium even in the absence of tight junctions.

Profiling of differentially expressed genes in porcine epithelial cells derived from periodontal ligament and gingiva by DNA microarray.

OBJECTIVE: It is unknown which genes are differentially expressed in cultured epithelial cells derived from the epithelial rests of Malassez (ERM) in periodontal ligament and oral gingival epithelium (OGE). This study analysed the different gene expression of OGE and ERM cells using a DNA microarray technique. DESIGN: Epithelial cells from ERM and OGE were isolated from porcine periodontal ligament and oral gingival epithelium. Each RNA sample extracted from the cells was reverse transcribed into cDNA and labelled with either cytidine 5-dUTP (Cy5) or cytidine 3-dUTP (Cy3). These labelled cDNA probes were then mixed and simultaneously hybridised to the Pig 13K microarray plate bearing 13,295 different genes (Operon, AL). Cellular enzyme-linked immunosorbent assay (CELISA) was performed to confirm the expression at protein level. RESULTS: There were nine genes common to the triplicate microarrays in ERM cells and one in OGE cells. Four of the nine genes including tissue factor (TF), FAT cadherin (FAT) and two unknown genes were expressed at levels more than threefold higher in ERM cells than in OGE cells. The protein levels of both TF and FAT in ERM cells were significantly higher than those in OGE. CONCLUSION: TF and FAT may act as markers to distinguish ERM cells from OGE cells in vitro.

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

BACKGROUND: Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis. METHODS: All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies. RESULTS: A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not. CONCLUSIONS: The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

Role of beta-defensins in oral epithelial health and disease.

The oral epithelium functions as a mechanical and protective barrier to resist bacterial infection. beta-Defensins are a group of antimicrobial peptides mainly produced by epithelial cells of many organs including skin, lung, kidney, pancreas, uterus, eye, and nasal and oral mucosa. This review focuses on beta-defensins (BDs) in oral epithelia and discusses their importance in oral epithelial health and disease. BDs exhibit antimicrobial activity against oral microbes including periodontitis-related bacteria, Candida, and papilloma virus. Alterative expression of BDs was observed in oral epithelial diseases, including oral inflammatory lesions with and without microbial infection and oral cancer. BDs may be useful in the treatment of oral infectious diseases, ulcerative lesions, and cancer. BDs play an important role in protection against oral microbes and may be used in clinical applications.

Expression pattern of p63 in oral epithelial lesions and submucous fibrosis associated with betel-quid chewing in Sri Lanka.

Betel-quid chewing, which is closely related to the high incidence of oral cancer, is prevalent in Sri Lanka. p63 has a remarkable structural similarity to p53, suggesting an aberrant expression in oral cancer. Using anti-p63 antibody and immunohistochemistry, the present study investigated the expression pattern of p63 in oral epithelial lesions, including different types of squamous cell carcinoma (SCC), different grades of epithelial dysplasia, and submucosal fibrosis associated with betel-quid chewing. Nuclear immunoreactivity for p63 was detected in all the cases, including normal oral epithelium and epithelial lesions. In normal oral epithelium, nuclear positivity for p63 was observed in some of the basal cell layers and focally in the parabasal layer. Nuclear positivity increased in the epithelial lesions. The percentage of positive nuclei in the epithelial lesions was significantly higher than in normal epithelium (P < 0.01) and was also significantly higher in oral submucosal fibrosis than in epithelial dysplasia (P < 0.05). The results indicate that the overexpression of p63 in oral precancerous lesions and SCC in betel-quid chewers in Sri Lanka may be a useful marker for oral precancerous lesions.

Increased expression of beta-defensin-2 and -3 during the development of autoimmune sialoadenitis in MRL/lpr mice.

beta-Defensins (BD) are small cationic antimicrobial peptides that produced principally in the epithelial cells of a number of organs. The present study analyzed the expression patterns of BD-1, -2, and -3 during the development of sialoadenitis in MRL/lpr mice. Submandibular glands were dissected from MRL/lpr mice at 4, 8, 10, 12, 14, and 16 weeks of age. The expression of mouse (m) BD-1, -2, and -3 mRNAs was examined by RT-PCR and quantified using TaqMan real-time RT-PCR. No significant differences in the level of expression of mBD-1 were observed among mice of different ages. The level of expression of mBD-2 was significantly higher at 16 weeks than at 4 or 8 weeks. The expression level of mBD-3 was highest in 14-week-old mice and was significantly higher than in 4-week or 16-week-old animals. Immunohistochemical staining showed that BD-3 was clearly localized in the ductal cells with variable intensities. In the center of the foci of lymphatic infiltration, ductal staining was faint or often not present. The results indicate that BD-2 and -3 may be upregulated during the development of autoimmune sialoadenitis.

Ameloblastic carcinoma ex ameloblastoma: report of a case-possible involvement of CpG island hypermethylation of the p16 gene in malignant transformation.

Ameloblastic carcinoma is described in the updated World Health Organization (WHO) classification as a rare malignant lesion. Ameloblastic carcinoma meeting the WHO criteria may arise either as a result of malignant change in a pre-existing benign ameloblastoma (carcinoma ex ameloblastoma) or as a primary malignant ameloblastoma not preceded by an ordinary ameloblastoma (de novo carcinoma). We report a case of ameloblastic carcinoma ex ameloblastoma and examine how this case underwent malignant transformation. The DNA was extracted separately from benign and malignant areas in paraffin sections of the tumor. Direct sequencing showed no genetic mutation of exons 5-8 of the p53 gene. Hypermethylation of CpG islands of the p16 gene was detected in the malignant parts of the tumor. The results indicate that hypermethylation of p16 may have been involved in the malignant transformation of the ameloblastoma in the present case.

Impaired alveolization in mice deficient in membrane-type matrix metalloproteinase 1 (MT1-MMP).

To clarify the involvement of membrane-type matrix metalloproteinase 1 (MT1-MMP) in lung organogenesis, we studied the lung morphology of 13-day-old MT1-MMP null mice. The lung architecture in MT1-MMP null mice was abnormal, and the airspace compartments were characterized by smooth walls and larger size. Most of the compartment wall consisted of one or two layers of cells and interstitial connective tissue that was thicker than that of normal alveoli. The wall frequently had capillaries on both sides of the interstitial connective tissue. These findings indicate that the lung in MT1-MMP null mice at 13 days of age is comparable to that of neonatal mice, i.e., it represents the stage before alveolization, suggesting that the generation of a large respiratory surface - the final process of lung development - is impaired in MT1-MMP null mice. Moreover, a zymography assay revealed decreased activity of matrix metalloproteinase 2 (MMP-2) in MT1-MMP null mice, suggesting that activation of pro-MMP-2 by MT1-MMP is critical in this process.

Effect of defensin peptides on eukaryotic cells: primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines.

BACKGROUND: Although the usefulness of the antimicrobial peptides known as defensins has been suggested against oral and skin infections, possible adverse effects of the defensins on the host should be understood before clinical applications can be contemplated. OBJECTIVE: In the present study, we investigated how alpha-defensin (HNP-1) and beta-defensins (hBD-1, -2, -3) affect cells including primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines, SCC-9 and KB. METHOD: Cell proliferation was assessed by the direct cell counting and XTT assay. RESULTS: We found that alpha-defensin promotes proliferation of the epithelial cells at low concentration but has a cytotoxic effect at high concentration. In contrast, beta-defensins have little effect on these cells at any concentration, suggesting that beta-defensins may have no adverse effects on the host. CONCLUSION: Therefore, in terms of host response beta-defensins may be more suitable antimicrobial agents for clinical applications than alpha-defensins.

Correlated expression of human beta defensin-1, -2 and -3 mRNAs in gingival tissues of young children.

OBJECTIVE: Human beta-defensins (hBDs) are a group of antimicrobial peptides, expressed by the epithelial cells of many organs including gingival epithelium. The present study examined correlation between the gene expressions of hBD-1, -2, -3 mRNAs and the inflammatory cytokines in human gingival tissues. STUDY DESIGN: The gingival tissues were obtained from surgical discards from 20 different patients (age range, 5-13 years). The expression levels of mRNAs were evaluated by quantitative RT-PCR with LightCycler. The mRNA expression levels were normalized with those of keratin 10 mRNA. The data were statistically analysed using Person's correlation coefficient. RESULTS: The expression levels of hBD-1,-2 and -3 were significantly correlated with each other and also correlated with that of TNF-alpha. CONCLUSIONS: The results indicate that the expression levels of hBDs vary from one individual to another.

Atypical carcinoid (neuroendocrine carcinoma) of the gingiva: counterpart of a laryngeal tumor.

Intraoral localization of neuroendocrine carcinoma, usually called Merkel cell carcinoma, is extremely rare. A case of neuroendocrine carcinoma that was a counterpart of laryngeal neuroendocrine carcinoma but was not a Merkel cell carcinoma, occurring at the mandibular gingiva in a 69-year-old Japanese man, is described. The tumor formed a cauliflower-like mass, measuring 20 x 20 mm, with a small area of necrosis. A computed tomography image showed metastasis in the right submandibular lymph node. Histopathologically, the tumor was composed of immature, small round cells that formed anastomosing trabecular nests. Few mitotic and no necrotic features were observed in the nests. Immunohistochemical studies showed positive staining for chromogranin, synaptophysin and neuron-specific enolase in the tumor nests. We diagnosed it as an atypical carcinoid (neuroendocrine carcinoma), a counterpart to the same type of tumor occurring in the larynx. The present case is an extremely rare case of neuroendocrine carcinoma without the feature of Merkel cell carcinoma arising from the gingiva.

Localization of human beta-defensin 3 mRNA in normal oral epithelium, leukoplakia, and lichen planus: an in situ hybridization study.

Human beta-defensin 3 (hBD-3), an antimicrobial peptide, is produced by various epithelial and some nonepithelial tissues. hBD-3 mRNA is widely expressed in oral tissues, including oral epithelium and the salivary glands. Although the localization of hBD-1 and hBD-2 has been well demonstrated in tissue sections, the localization pattern of hBD-3 has not yet been shown. In the present study, we investigated the expression pattern of hBD-3 mRNA by in situ hybridization using specific RNA probes; the signal for hBD-3 was detected in upper spinous and granular layers in normal oral epithelium. In cases of leukoplakia, a strong signal of hBD-3 mRNA was observed in the granular layer. In lichen planus, the signal was strongly detected in the spinous and suprabasal layers. The signals were stronger than those of either normal oral epithelium or leukoplakia. The results indicate that the localization pattern of hBD-3 is very similar to that of hBD-2. hBD-2 and hBD-3 may function together or compensate each other for expressional loss.

Solcoseryl, a tissue respiration stimulating agent, significantly enhances the effect of capacitively coupled electric field on the promotion of bone formation around dental implants.

In the present study we examined the combined effect of application of a capacitively coupled electric field (CCEF) and the tissue respiration stimulating agent, Solcoseryl, on the promotion of bone formation around dental implants histologically and mechanically. After a dental implant was inserted into each femur of Japanese white rabbits, Solcoseryl (2 ml/kg) was administered intravenously in the ear vein and a CCEF was applied for 4 h per day for 14 days. The degree of bone formation on microscopic observation, bone contact ratio, bone surface area ratio, and the level of removal torque of the implant in the Solcoseryl- and CCEF-treated group were significantly higher than the respective value in the control group, which had not been treated with Solcoseryl nor CCEF. Thus, the combination of CCEF stimulation and Solcoseryl effectively promoted the formation of new bone. It is suggested that the clinical use of a combination of CCEF stimulation and Solcoseryl for dental implants promotes osseointegration.

Upregulated expression of human beta defensin-1 and -3 mRNA during differentiation of keratinocyte immortalized cell lines, HaCaT and PHK16-0b.

BACKGROUND: Human beta-defensins (hBDs) belong to a group of antimicrobial peptide that are expressed in the epithelial cells. OBJECTIVE: The present study investigated mRNA expression levels of the beta-defensins, hBD-1, -2 and -3, in human keratinocytes during differentiation in vitro. METHODS: Immortalized keratinocyte cell lines, HaCaT and PHK16-0b, were used in this study; in order to stimulate differentiation, the Ca(2+) concentration in the growth media was increased from 0.3 to 1.8 mM. RESULTS: Four days after the increase, the expression levels of hBD-1 and -3 were increased in both cell lines, followed by an increase in the mRNA levels of the differentiation markers, involucrin and keratin 10. No increased expression of hBD-2 was observed. CONCLUSION: The results indicate that keratinocyte differentiation may stimulate hBD-1 and -3 expression in stratified squamous epithelia.

Expression of MIP-3alpha/CCL20, a macrophage inflammatory protein in oral squamous cell carcinoma.

We have examined the expression of MIP-3alpha/CCL20 in oral squamous cell carcinoma (SCC) in vivo and in vitro. In addition, we have investigated whether the expression of MIP-3alpha/CCL20 is regulated by bacterial infection and inflammatory cytokines. In order to determine the mRNA level of MIP-3alpha, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with LightCycler using the double-stranded DNA dye, SYBR Green I. Oral epithelial cells and six SCC cell lines (SCC-9, SAS, BSC-OF, HSC-4, HSC, Ca9-22) were found to express MIP-3alpha mRNA. The expression of MIP-3alpha was upregulated by infection with Actinobacillus actinomycetemcomitans and by stimulation with lipopolysaccharide and TNF-alpha. By in situ hybridization, the detectable MIP-3alpha expression in SCC was localized primarily at the epithelial pearls corresponding to the spinous layer. These results suggest that MIP-3alpha contributes to the oral immunoresponse to bacterial infection, and may be involved in the growth of SCC.

Ultrastructural study of synovitis induced by trauma to the rat temporomandibular joint (TMJ).

BACKGROUND: Electron microscopy was used to examine the histologic effect of trauma on the rat temporomandibular joint synovial membrane. METHODS: Trauma to the TMJ in male Wister rats (100-200 g) was introduced through repeated forced condylar hypermobility. Ultrastructural observations were made 5 days and 6 weeks after the trauma. RESULTS: The early response of the synovial membrane was synovial hyperplasia, type A synovial cell loss, dilation of the r-ER in the type B synovial cells and fibrin deposition on the synovial surfaces. The late response included degeneration of synovial cells with swollen mitochondria and cell projections, and cell fragmentation. Large amount of fibrin deposition on opposing surface layers was also noticed. CONCLUSION: The type A cell loss and fibrin deposition followed by the occurrence of fibrinous materials at opposing surface layers of the synovial membrane suggest that traumatic synovitis causes synovial adhesions.

Defensins in saliva and the salivary glands.

Saliva contains several types of antimicrobial peptides that play a role in innate immunity. Peptides that were recently added to this list are the defensins. The purpose of this review is to summarize what is known about the production and role of defensins in the salivary glands and to discuss their therapeutic potential. The Alpha-defensins, human neutrophil defensins (HNP)-1, -2, and -3, have been detected in saliva and may be derived from neutrophils. The Beta-defensins, human Beta-defensins (HBD)-1 and -2, have also been detected in saliva. Although it has been speculated that salivary HBDs are derived from keratinocytes that line the oral mucosa rather than from the salivary glands, the HBD-1 peptide was recently found to be specifically expressed in salivary ductal cells, although not in acini. Defensins may be useful for the treatment of periodontal disease and for the prevention of caries and periodontitis.

Upregulation of human beta-defensin 2 peptide expression in oral lichen planus, leukoplakia and candidiasis. an immunohistochemical study.

Human beta defensin 2 (hBD-2) is a major antimicrobial peptide that is produced by many types of epithelial cells, and is transcriptionally inducible by various proinflammatory agents, such as cytokines and bacteria. Although in vitro studies of the hBDs in oral epithelial cells have been well documented, only little is known about the in vivo pathological state of oral epithelium. We investigated the localization of hBD-2 peptide in tissue sections of oral lichen planus, leukoplakia, candidal leukoplakia and radicular cysts using immunohistochemistry. HBD-2 was stained in both the hyperkeratinized and the granular layers in cases of lichen planus with hyperkeratosis and leukoplakia. Expression in spinous and suprabasal layers was often strong in lichen planus. There were no significant differences in the number of S-100 positive dendritic cells between the widely stained areas and those with limited staining areas in lichen planus. In cases of candidal leukoplakia, the hyphae of candida were mainly detected on the surface of keratinization, which showed only negative or faint staining for hBD-2. These results suggest that hBD-2 is vigorously induced by lichen planus-related inflammation and that it plays an important role in protection from Candida albicans infection; however, it is not a strong chemotactic attractant for Langerhans cells in pathological conditions of oral epithelium.