Hmb-45 May Be a More Sensitive Maker Than S-100 or Melan-a for Immunohistochemical Diagnosis of Primary Oral and Nasal Mucosal Melanomas

Background: Primary mucosal melanomas (MMs) of the head and neck are a rare entity. Melanomas with characteristic melanin-pigmented tumor cells are easy to diagnose, but those without melanin-pigmented tumor cells, amelanotic melanomas, are difficult to identify and need immunohistochemistry (IHC) to confirm the final diagnosis. In this study, we examined the expression of three melanocytic differentiation markers, HMB-45, S-100, and Melan-A in primary oral and nasal MMs. We tried to evaluate whether HMB-45, S-100, and Melan-A were useful for diagnosis of primary oral and nasal MMs and to find out which marker was the best of the three. Methods: This study used IHC to examine the expression of HMB- 45, S-100, and Melan-A in 17 formalin-fixed paraffin-embedded specimens of primary oral and nasal MMs. The staining intensities (SIs) and labeling indices (LIs) of HMB-45, S-100, and Melan-A in 17 MMs were calculated and compared between any two markers. Results: Immunostaining results showed that the positive rate was 94% (16 of 17) for HMB-45, 88% (15 of 17) for S -100, and 71% ( 12 of 17) for Melan-A in 17 MMs. The SI of HMB-45 was significantly higher than that of S-100 (P = 0.0011) or of Melan-A (P = 0. 0034). In addition, the mean LI of Melan-A ( 59 ± 43%) was significantly lower than that of HMB-45 (83±28%, P = 0.0065) or of S-100 (79 ± 33%, P = 0.0237). Conclusions: Our results indicate that both HMB-45 and S-100 show a high positive rate and LI in MMs and therefore may be good markers for immunohistochemical diagnosis of primary oral and nasal MMs. In addition, HMB-45 may be a more sensitive marker than S-100 because HMB-45 shows a significantly higher SI than S-100 in this study

Comparison of Nd:Yag Laser Versus Scaling and Root Planing in Periodontal Therapy

BACKGROUND: The Nd:YAG laser has recently been used in the treatment of periodontal disease. However, although a clinical reduction of probing depth and gingival inflammation to this new approach has been reported, it has not been fully evaluated. Interleukin-1 beta (IL- 1beta), a potent stimulator of bone resorption, has been identified in gingival crevicular fluid (GCF), which is closely associated with periodontal destruction. The aim of this study was to compare the effects of Nd:YAG laser treatment versus scaling/root planing (SRP) treatment on crevicular IL -1beta levels in 52 sampled sites obtained from 8 periodontitis patients. METHODS: One or 2 periodontitis- affected sites with a 4 to 6 mm probing depth and horizontal bone loss from 3 adjacent single-root teeth in each of 4 separate quadrants were selected from patients for clinical documentation and IL-1beta assay. Sampling site(s) from each diseased quadrant was randomly assigned to one of the following groups: 1) subgingival laser treatment (20 pps, 150 mJ) only; 2) SRP only; 3) laser treatment first, followed by SRP 6 weeks later; or 4) SRP first, followed by laser therapy 6 weeks later. The GCF was collected and the amount of IL-1beta was assayed by enzyme-linked immunosorbent assay (ELISA). Clinical parameters and GCF were measured at baseline and biweekly after therapy for 12 weeks. RESULTS: An obvious clinical improvement (marked decrease in the number of diseased sites with gingival index > or =2) and reduction of crevicular IL- 1beta were found in all groups. The level of IL- 1beta was significantly lower in the SRP group (P = 0.035) than in the laser therapy group for the duration of the 12 weeks. The laser combined SRP therapy group showed a further reduction of IL- 1beta (6 to 12 weeks after treatment) than either laser therapy alone or SRP combined laser therapy. CONCLUSIONS: Our data suggest that laser therapy appeared to be less effective than traditional SRP treatment. Of the 4 treatment modalities, inclusion of SRP was found to have a superior IL- 1beta response, when compared to other therapies without it. In addition, no additional benefit was found when laser treatment was used secondary to traditional SRP therapy

In Vitro Effect of Laser Irradiation on Cementum-Bound Endotoxin Isolated from Periodontally Diseased Roots

Background: In a previous study, we evaluated the in vivo effects of an Nd:YAG laser on periodontal disease by measuring crevicular interleukin ( IL)1beta levels before and after laser application. It was found that laser therapy was less effective than traditional scaling and root planing . These results might be due to incomplete removal of microbial residues and cementum-bound endotoxin on root surfaces by the laser. In this study, we explored the in vitro effectiveness of an Nd:YAG laser for the elimination of cementum-bound endotoxin by measuring ILAP changes in stimulated monocytes. Methods: Fresh human monocytes were harvested from adults without periodontitis and grown in RPMI 1640 medium. Diseased cementum particles were collected and prepared from teeth with untreated periodontitis and were irradiated with 5 levels of laser energy. Cementum particles were subjected to endotoxin testing by a limulus amebocyte lysate (LAL) assay and then were incubated with cultured monocytes. Production of IL-1beta in stimulated monocytes was measured by enzyme- linked immunosorbent assay and quantified by spectrophotometry. Results: The endotoxin unit (EU) of diseased cementurn was 18.4 EU/mg, which seemed to be remarkably lower than that of common periodontal pathogens including Porphyromonas gingivalis (381) at 15,300 EU/mg/ml, Prevotella intermedia (ATCC 25611) at 227 EU/mg/ ml, and Fusobacterium nucleatum ( ATCC 25586) at 1,987 EU/mg/ ml. Monocytes subjected to stimulation by diseased cementurn particles without laser irradiation produced 124 to 145 pg/ ml IL-1beta, 9- to 18-fold higher than that of unstimulated monocytes (7.07 to 15.95 pg/ml). Diseased cementum particles after irradiation with various energy levels of the Nd:YAG laser could still stimulate monocytes to secrete 89 to 129 pg/ml IL-1beta. No statistically significant difference was found in the production of IL-1 beta induced by diseased- bound cementurn with or without laser irradiation. Conclusions: The Nd:YAG laser varying from 50 mJ, 10 pps to 150 mJ, 20 pps, for 2 minutes, did not seem to be effective in destroying diseased cementurn endotoxin

Association of pocket epithelial cell proliferation in periodontitis with TLR9 expression and inflammatory response

Inflammatory response is triggered after recognition of microbial ligands by innate receptors such as Toll-like receptors (TLRs) and Nucleotide oligomerization domain (NOD)-like receptors (NLRs). In this study, we examined serial frozen sections of gingival biopsies from patients with gingivitis or periodontitis by immunohistochemical analysis for the topographic expression patterns of selected innate receptors and their association with cell proliferation in clinically healthy and diseased gingival tissues. Methods: A total of 19 gingival biopsies were collected from patients at the School of Dentistry, National Taiwan University Medical Center according to approved protocol and with informed consent. The specimens were assigned to either the gingivitis group or periodontitis group after clinical evaluation using gingival index. Frozen sections of gingival biopsies were stained with hematoxylin and eosin for histological evaluation. Serial sections of the same samples were stained with a panel of antibodies for immunohistochemical analysis. Expression of each protein marker was compared in the oral versus the sulcular epithelium of the same section. Results: Expression of cytokeratin 19 (CK19) was markedly increased in the basement membranes of the oral epithelium and in all layers of the pocket epithelium where it caused evident cell proliferation and migration of sulcular epithelial cells into the lamina propria of periodontitis tissue. TLR4 and the cytoplasmic NLRP3 were expressed in all sections examined regardless of disease state. However, expression of TLR9-, CK19- and collagenolytic matrix metalloproteinase-13 and activated NF-κB subunit p65 was more commonly found in periodontitis tissues than in gingivitis tissues. Conclusion: Activation of TLR9 signaling in the pocket epithelium was highly associated with periodontal inflammation and possibly with loss of tissue integrity. Further studies of mechanisms by which TLR9 signaling is activated in the periodontal epithelium may lead to new strategies for treating periodontitis

Association of initial mucogingival status with clinical outcome of non-surgical periodontal therapy: A retrospective analysis of 204 patients

Background/Purpose: This study was conducted to evaluate the influence of mucogingival parameters, including keratinized mucosa (KM) and attached gingiva (AG), on the outcome of non-surgical periodontal therapy (NSPT). Methods: A total of 204 non-smoking patients with generalized chronic periodontitis who received NSPT between 2012 and 2014 were included. The Mantel–Haenszel chi-square test was used to assess the associations between initial mucogingival parameters and initial clinical parameters on the buccal aspect, and the associations between initial mucogingival parameters and outcome clinical parameters on the buccal aspect of the sites with severe periodontal destruction. The generalized liner model was used to evaluate the contribution of initial clinical parameters to the outcome of NSPT. Results: KM ≥ 3 mm was associated with greater probing pocket depth (PD), less gingival recession (REC), and less clinical attachment level (CAL), and AG < 1 mm was associated with greater PD, REC, and CAL before NSPT. At the sites with severe periodontal destruction, KM ≥ 3 mm was associated with greater PD reduction (0.25 ± 0.08 mm) and CAL gain (0.25 ± 0.09 mm), and AG < 1 mm was associated with greater CAL gain (0.15 ± 0.08 mm) after NSPT. Initial PD ≥ 7 mm and non-molar teeth showed greater contribution to the outcome of NSPT. Conclusion: Less AG (<1 mm) was associated with greater periodontal destruction at baseline. At the sites with severe periodontal destruction, greater KM (≥3 mm) and less AG (<1 mm) resulted in better outcomes of NSPT. Keywords: Gingiva, Periodontitis, Root planing, Non-surgical periodontal therap

Epigallocatechin-3-gallate inhibits lysophosphatidic acid-stimulated connective tissue growth factor via JNK and Smad3 suppression in human gingival fibroblasts

Connective tissue growth factor (CTGF/CCN2) is involved in the development and progression of fibrotic diseases, including gingival overgrowth (GO). Recent studies indicate that lysophosphatidic acid (LPA) is also significantly involved in wound healing and the development of fibrosis. This study investigated whether epigallocatechin-3-gallate (EGCG) can inhibit LPA-induced CCN2 expression in human gingival fibroblast (GF) and its mechanism. Methods: Western blot analyses were used to study the signaling pathways of LPA-induced CCN2 expression in human GFs and the effects of EGCG on this pathway. Results: LPA stimulated CCN2 synthesis in human GFs. This effect can be significantly inhibited bytransforming growth factor-β type I receptor/ALK5, Smad3, and JNK inhibitors but not ERK, P38, and MAPK inhibitors. EGCG completely inhibited LPA-induced CCN2 expression through attenuating the LPA-induced JNK and Smad3 phosphorylation in human GFs. Conclusion: LPA produced at the surgical wound may contribute to the recurrence of GO by upregulating CCN2 expression in human GFs. This effect was mediated by Smad3 and JNK activation and ALK5 transactivation. EGCG could be a useful agent for reducing the recurrence of GO after surgery through suppression of JNK and Smad3 activations

Sphingosine-1-phosphate stimulated connective tissue growth factor expression in human buccal fibroblasts: Inhibition by epigallocatechin-3-gallate

Connective tissue growth factor (CCN2) has been associated with the pathogenesis of various fibrotic diseases, including oral submucous fibrosis (OSF). The chemical constituents of areca nut along with the mechanical trauma cause OSF. The coarse fibers of areca nut injure the mucosa and hence sphingosine-1-phosphate (S1P) is released at the wounded sites. Recent studies have shown that S1P is involved in wound healing and the development of fibrosis. The aims of this study were to investigate the effects of S1P on CCN2 expression in human buccal fibroblasts (HBFs) and identify the potential targets for drug intervention or chemoprevention of OSF. Methods: Western blot analyses were used to study the effects of S1P on CCN2 expression and its signaling pathways in HBFs and whether epigallocatechin-3-gallate (EGCG), the main and most significant polyphenol in green tea, could inhibit this pathway. Results: S1P significantly enhanced CCN2 synthesis in HBFs. This effect can be inhibited by c-Jun NH2-terminal kinase (JNK) inhibitor and extracellular signal-regulated kinase inhibitor but not by P38 mitogen-activated protein kinase inhibitor. Interestingly, EGCG completely blocked S1P-induced CCN2 expression via suppressing S1P-induced JNK phosphorylation. Conclusion: S1P released by repetitive mechanical trauma during AN chewing may contribute to the pathogenesis of OSF through upregulating CCN2 expression in HBFs. EGCG could be an adjuvant to the current offered therapy options or the prevention of OSF through suppression of JNK activation