Effects of Low-Level Laser Therapy on Collagen Expression and Neutrophil Infiltrate in 5-Fluorouracil-Induced Oral Mucositis in Hamsters

Background and Objectives: Several studies have suggested that low-level laser therapy (LLLT) can ameliorate oral mucositis, however, the mechanisms involved are not well understood. The aim of this study was to investigate the mechanisms of action of LLLT on chemotherapy-induced oral mucositis, as related to effects on collagen expression and inflammation Materials and Methods: A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil, followed by surface irritation. Animals were randomly allocated into three groups, and treated with an InGaAIP diode laser at a wavelength of 660 nm and output power of 35 or 100 mW laser, or no laser Clinical severity of mucositis was assessed at four time-points by a blinded examiner Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. Collagen was qualitatively and quantitatively evaluated after picrosinus staining. The density of the neutrophil infiltrate was also scored Results: Peak clinical severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW and control groups The reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a decrease in the number of neutrophils and an increase in the proportion of mature collagen as compared to the other two groups. The total quantity of collagen was significantly higher in the control (no laser) group at the day 11 time-point, as compared to the 35 mW laser group, consistent with a more prolonged inflammatory response in the control group. Conclusion: This study supports two mechanisms of action for LLLT in reducing mucositis severity. The increase in collagen organization in response to the 35 mW laser indicates that LLLT promotes wound healing In addition, LLLT also appears to have an anti-inflammatory effect, as evidenced by the reduction in neutrophil infiltrate Lasers Surg Med 42 546-552, 2010. (C) 2010 Wiley-Liss, Inc.

Cyclooxygenase-2 and vascular endothelial growth factor expression in 5-fluorouracil-induced oral mucositis in hamsters: evaluation of two low-intensity laser protocols

The aim of this study was to investigate the mechanisms whereby low-intensity laser therapy may affect the severity of oral mucositis. A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil followed by surface irritation. Animals were randomly allocated into three groups and treated with a 35 mW laser, 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. This tissue was used for immunohistochemistry for cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and factor VIII (marker of microvessel density) and the resulting staining was quantified. Peak severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW laser and control groups. This reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a significantly lower level of COX-2 staining. The 100 mW laser did not have an effect on the severity of clinical mucositis, but was associated with a decrease in VEGF levels at the later time-points, as compared to the other groups. There was no clear relationship of VEGF levels or microvessel density to clinical mucositis severity. The tissue response to laser therapy appears to vary by dose. Low-intensity laser therapy appears to reduce the severity of mucositis, at least in part, by reducing COX-2 levels and associated inhibition of the inflammatory response.

Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

Introduction: The objective of this study was to investigate the expression of matrix metalloproteinases (MM Ps) in apical periodontitis and during the periapical healing phase after root canal treatment. Methods: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. Results: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusions: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes. (J Endod 2010;36:231-237)

Endodontic Chelators Induce Nitric Oxide Expression by Murine-cultured Macrophages

Introduction: Endodontic chelators may extrude to apical tissues during instrumentation activating cellular events on periapical tissues. This study assessed in vitro the expression of nitric oxide (NO) concentrations by murine peritoneal macrophages after contact with MTAD (Dentsply/Tulsa, Tulsa, OK), Tetraclean (Ogna Laboratori Farmaceutici, Muggio, Italy), Smear Clear (Sybron Endo, Orange, CA), and EDTA (Biodinamica, Ibipora, PR, Brazil). Methods: Macrophage cells were obtained from Swiss mice after peritoneal lavage. Chelators were diluted in distilled water obtaining 12 concentrations, and MTT assay identified the concentrations, per group, displaying the highest cell viability (analysis of variance, p < 0.01). Selected concentrations were tested for NO expression using Griess reaction. Culture medium and lipopolysaccharide (LPS) were used as controls. Results: Analysis of variance and Tukey tests showed that all chelators displayed elevated NO concentrations compared with the negative control (p < 0.01). MTAD induced the lowest NO expression, followed by Tetraclean, EDTA, and Smear Clear. No difference was observed between MTAD and Tetraclean (p > 0.01), Tetraclean and EDTA (p > 0.01), and EDTA and Smear Clear (p > 0.01). LPS ranked similar to both EDTA and Smear Clear (p > 0.01). Conclusion: The tested endodontic chelators displayed severe proinflammatory effects on murine-cultured macrophages. Citric acid-based solutions induce lower No release than EDTA-based irrigants. (J Endod 2009;35:824-828)

Expression of metallothionein in ameloblastoma. A regulatory molecule?

BACKGROUND: Ameloblastoma is a benign odontogenic tumor, exhibiting local invasiveness and high rate of recurrence. Metallothionein is a protein associated with tumorigenesis, serving as prognostic factor in different neoplasms. We are interested in mechanisms underlying ameloblastoma local invasiveness. Thus, we decided to analyze expression of metallothionein in this tumor. MATERIALS AND METHODS: An immunohistochemical evaluation of metallothionein in ameloblastoma was carried out. As control, we assessed expression of the same molecule in calcifying cystic odontogenic tumor (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium. RESULTS: We studied 12 cases of solid/multicystic ameloblastomas. Metallothionein was observed in all samples. This molecule was observed in columnar cells in the periphery and in central polyhedral cells. CCOT (four cases) also showed the presence of metallothionein. Morphometry of stained areas showed that expression of metallothionein in ameloblastoma was significantly higher compared to CCOT (P < 0.0001). CONCLUSIONS: This protein may have an impact on ameloblastoma behavior. Metallothionein would act as a zinc reservoir for important proteases related to ameloblastoma biology, such as MMPs. This protein could also display pro-mitotic and anti-apoptotic features in the tumor. J Oral Pathol Med (2011) 40: 516-519

Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women

Hyperglycemia occurs in a variety of conditions such as overt diabetes, gestational diabetes and mild hyperglycemia, all of which are generally defined based on the oral glucose tolerance test and glucose profiles. Whereas diabetes has received considerable attention in recent decades, few studies have examined the mechanisms of mild hyperglycemia and its associated disturbances. Mild gestational hyperglycemia is associated with macrosomia and a high risk of perinatal mortality. Morphologically, the placenta of these women is characterized by an increase in the number of terminal villi and capillaries, presumably as part of a compensatory mechanism to maintain homeostasis at the maternal-fetal interface. In this study, we analised the expression of VEGF and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in placentas from mildly hyperglycemic women. This expression was compared with that of normoglycemic women and women with gestational and overt diabetes. Immunohistochemistry revealed strong staining for VEGF and VEGFR-2 in vascular and trophoblastic cells of mildly hyperglycemic women, whereas the staining for VEGFR-1 was discrete and limited to the trophoblast. The pattern of VEGF and VEGF-receptor reactivity in placentas from women with overt diabetes was similar to that of normoglycemic women. In women with gestational diabetes, strong staining for VEGFR-1 was observed in vascular and trophoblastic cells whereas VEGF and VEGFR-2 were detected only in the trophoblast. The expression of these proteins was confirmed by western blotting, which revealed the presence of an additional band of 75 kDa. In the decidual compartment, only extravillous trophoblast reacted with all antibodies. Morphological analysis revealed collagen deposition around large arteries in all groups with altered glycemia. These findings indicate a placental response to altered glycemia that could have important consequences for the fetus. The change in the placental VEGF/VEGFR expression ratio in mild hyperglycemia may favor angiogenesis in placental tissue and could explain the hypercapillarization of villi seen in this gestational disturbance. (C) 2010 Elsevier Ltd. All rights reserved.

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and beta 1 integrin

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha 1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha 1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73induced colocalization of syndecan-1 and beta 1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha 1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta 1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

Association of Human T Lymphotropic Virus 1 Amplification of Periodontitis Severity with Altered Cytokine Expression in Response to a Standard Periodontopathogen Infection

Background. Periodontal diseases (PDs) are infectious diseases in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. Recently, viruses have been implicated in the pathogenesis of PDs. Individuals infected with human T lymphotropic virus 1 (HTLV-1) present with abnormal oral health and a marked increased prevalence of periodontal disease. Methods. In this study, we investigated the patterns of periodontopathogen infection and local inflammatory immune markers in HTLV-1-seropositive individuals with chronic periodontitis (CP/HTLV-1 group) compared with HTLV-1 -seronegative individuals with chronic periodontitis (CP group) and periodontally healthy, HTLV-1 -seronegative individuals (control group). Results. Patients in the CP/HTLV-1 group had significantly higher values of bleeding on probing, mean probing depth, and attachment loss than patients in the CP group. The expression of tumor necrosis factor a and interleukin (IL) 4 was found to be similar in the CP and CP/HTLV-1 groups, whereas IL-12 and IL-17 levels trended toward a higher expression in the CP/HTLV-1 group. A significant increase was seen in the levels of IL-1 beta and interferon gamma in the CP/HTLV-1 group compared with the CP group, whereas expression of the regulatory T cell marker FOXp3 and IL-10 was significantly decreased in the lesions from the CP/HTLV-1 group. Interestingly, similar frequency and/or load of periodontopathogens (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans) and frequency of viruses (herpes simplex virus 1, human cytomegalovirus, and Epstein-Barr virus) characteristically associated with PDs were found in the CP/HTLV and CP groups. Conclusions. HTLV-1 may play a critical role in the pathogenesis of periodontal disease through the deregulation of the local cytokine network, resulting in an exacerbated response against a standard periodontopathogen infection.

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance ( HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting ( 72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology. ( Endocrinology 149: 6326-6335, 2008)