Effects of photobiomodulation on different phases of in vitro osteogenesis.

The present study aimed to evaluate the effect of photobiomodulation therapy (PBM) on different stages of osteogenesis in vitro. For this, osteoblastic-like cells (Saos-2 cell lineage) were irradiated in two different periods: during the Proliferation phase (PP; from the second to the fourth day) and during the Differentiation phase (DP; from the seventh to the ninth day). The energy density used in the study was 1.5 J/ cm2. The following parameters were evaluated: 1) quantification of collagen type 1 (COL 1), osteopontin (OPN), and bone morphogenetic protein 2 (BMP-2); 2) quantification of alkaline phosphatase (ALP) activity; and 3) quantification of  extracellular matrix (ECM) mineralization. Non-irradiated cultures were used as controls. The data were analyzed using the Student's t-test or one-way ANOVA, considering a significance level of 5%. The results indicated that COL 1 and BMP-2 quantification was higher in Saos-2 irradiated during the DP in relation to the control group at day 10 (p < 0.05). No differences were observed for other comparisons at this time point (p > 0.05). OPN expression was greater in PP compared with the other experimental groups at day 10 (p < 0.05). Irradiation did not affect ALP activity in Saos-2 regardless of the exposure phase and the time point evaluated (p > 0.05). At day 14, ECM mineralization was higher in Saos-2 cultures irradiated during the DP in relation to the PP (p < 0.05). In conclusion, the results suggested that the effects of PBM on osteoblastic cells may be influenced by the stage of cell differentiation.

Adenomatoid odontogenic tumor: Features of ameloblastic-like epithelial cells differentiation, secretion, and the nature of tumor cells products.

BACKGROUND: This study aimed to investigate the differentiation of ameloblastic-like cells and the nature of the secreted eosinophilic materials in adenomatoid odontogenic tumors. METHODS: We studied histological and immunohistochemical characteristics of 20 cases using: cytokeratins 14 and 19, amelogenin, collagen I, laminin, vimentin, and CD34. RESULTS: Rosette cells differentiated into ameloblastic-like cells positioned face-to-face, displaying collagen I-positive material between them. Epithelial cells of the rosettes can differentiate into ameloblastic-like cells. This phenomenon probably occurs due to an induction phenomenon between these cells. The secretion of collagen I is probably a brief event. Amelogenin-positive areas were interspersed by epithelial cells in the lace-like areas, outside the rosettes and distant from the ameloblastic-like cells. CONCLUSIONS: There are at least two types of eosinophilic material in different areas within the tumor, one in the rosette and solid areas and another in lace-like areas. The secreted eosinophilic material in the rosettes and solid areas is probably a product of well-differentiated ameloblastic-like cells. It is positive for collagen I and negative for amelogenin, whereas some eosinophilic materials in the lace-like areas are positive for amelogenin. We hypothesize that the latter eosinophilic material could be a product of odontogenic cuboidal epithelial or intermediate stratum-like epithelial cells.

Oral epithelial dysplasia grading: Comparing the binary system to the traditional 3-tier system, an actuarial study with malignant transformation as outcome.

BACKGROUND: Establishing the risk of malignant transformation (MT) in oral leukoplakia is usually based on grading oral epithelial dysplasia (OED) on biopsy tissue, for which two systems are proposed: a 3-tier and a binary system. Only very few actuarial studies have tested the accuracy of such methods in predicting MT, especially for the binary system. This study aimed to assess the accuracy of the two grading systems in predicting MT in a cohort of oral leukoplakia (OL) from Brazil, with follow-up data. METHODS: The sample comprised 878 individuals diagnosed with OL from 2005 to 2018. Follow-up data were obtained both locally and from the regional cancer registry. All lesions were graded using both the 3-tier and the binary systems. Kaplan-Meier curves (Log-rank Mantel-Cox) were used to assess risk and kappa to assess interobserver agreement. RESULTS: Thirty-five individuals underwent MT (4%). Both systems demonstrated prognostic value, though the 3-tier system proved superior, with OR 9.23 (3.42-23.69), PPV 0.152, NPV 0.98, compared to binary OR 3.49 (1.79-6.79), PPV 0.079, NPV 0.976. Interobserver agreement was also superior in the 3-tier system (0.47, p < 0.05) compared to the binary system (0.139, p = 0.39). Combining the two systems enhanced prognostic values (OR 14.28, PPV 0.217, NPV 0.981). CONCLUSION: The 3-tier system presented superior prognostic value to the binary system. Combining both systems to double-grade intermediate lesions might enhance risk assessment.

Microvesicles derived from squamous cell carcinoma induce cell death, autophagy, and invasion of benign myoepithelial cells.

BACKGROUND: There has been great interest recently in the mechanisms of cell-to-cell communication through microvesicles (MV). These structures are produced by many different cell types and can modulate cellular activity by induction of epigenetic alterations. These vesicles may promote tumor mass increase either by stimulating cell proliferation via growth factors or by inhibiting apoptosis, which reinforces the role of such vesicles as important modulators of tumor progression. METHODS: The present in vitro study aimed to characterize MV derived from malignant neoplastic epithelial cell cultures (EP) and their effect on the expression of apoptosis/autophagy and invasion related genes of benign myoepithelial (Myo) cell cultures. RESULTS: The results revealed round structures with a mean size of 153.6 (±0.2) nm, with typical MV morphology. CD63 quantification indicated that EP cell culture at 70%-80% confluence secreted 3.088 × 108 MV/mL. Overall, Myo exposed to MVs derived from EP showed both up- and downregulation of tumorigenesis promoting genes. MVs from EP cells promoted cell death of Myo cells and positively modulate BAX, SURVIVIN, LC3B, MMP-2, and MMP-9 expression. Furthermore, an increasing of MMP-2 and MMP-9 secretion by Myo was observed after MV exposure. CONCLUSIONS: These findings suggest that MVs from EP modulate autophagy of Myo cells, which may, in part, explain the disappearance of these cells in in situ areas of invasive carcinoma ex-pleomorphic adenoma. Additionally, the overexpression of MMPs contributes to the development of an invasive phenotype of Myo cells, which could favor the dissolution of the basement membrane during tumorigenesis process.

Effects of increasing nitrogen levels in Mombasa grass on pasture characteristics, chemical composition, and beef cattle performance in the humid tropics of the Amazon.

Improving beef cattle production in pasture-based systems without expanding agricultural land has been the focus of several studies over the last decades. Nitrogen fertilization is one of the available strategies with high potential to optimize cattle performance in tropical systems. This study aimed to evaluate the effects of increasing nitrogen levels (150, 300, and 450 kg ha-1 of N year-1) in Megathyrsus maximus cv. Mombasa in the humid tropics of the Amazon. The following parameters were evaluated: pasture agronomic characteristics, herbage chemical composition, and beef cattle performance. A total of 24 nine-month-old Nellore bulls with initial mean weight of 173 ± 1.95 kg were used for the performance test. The experimental design was a randomized block with three treatments. Herbage mass and leaf mass contents were increased by the highest nitrogen level (P < 0.05). Nitrogen levels elevated the forage accumulation rate, final leaf blade length, and pre-grazing canopy height(P < 0.05). The crude protein content increased at the highest N level (P < 0.05). The neutral detergent fiber and acid detergent fiber contents decreased with increasing N levels. The stocking rate and total weight gain per hectare were higher in bulls grazing pastures fertilized with 450 compared with 150 kg ha-1 of N year-1(P < 0.05). The average daily gain was influenced by N levels (P < 0.05). Nitrogen levels at 300 and 450 kg increased animal weight gain and productivity per area by increasing forage yield and improving the chemical composition of Mombasa grass.

MiR-455-3p, miR-150 and miR-375 are aberrantly expressed in salivary gland adenoid cystic carcinoma and polymorphous adenocarcinoma.

BACKGROUND: Adenoid cystic carcinoma (AdCC) and polymorphous adenocarcinoma (PAC) are included among the most common salivary gland cancers. They share clinical and histological characteristics, making their diagnosis challenging in specific cases. MicroRNAs (miRNA) are short, non-coding RNA sequences of 19-25 nucleotides in length that are involved in post-transcriptional protein expression. They have been shown to play important roles in neoplastic and non-neoplastic processes and have been suggested as diagnostic and prognostic markers. METHODS: This study, using quantitative RT-PCR, investigated miR-150, miR-455-3p and miR-375 expression, in order to identify a possible molecular distinction between AdCC and PAC. RESULTS: miRNA-150 and miRNA-375 expression was significantly decreased in AdCC and PAC compared with salivary gland tissue controls, whilst miRNA-455-3p showed significantly increased expression in AdCC when compared to PAC, (P < 0.05). miR-150, miR-357 and miR-455-3p expression in AdCC, PAC and control was not associated with age, gender nor with anatomic site (major and minor salivary glands) (P > 0.05). CONCLUSION: MiR-455-3p could be used as a complimentary tool in the diagnosis of challenging AdCC cases.

Epithelial membrane antigen and DOG1 expression in minor salivary gland tumours.

Epithelial membrane antigen (EMA) and DOG1 are used as marker of epithelial cells, particularly the luminal cells, of salivary gland tumours. The aim of this study was to compare the EMA and DOG1 expression in tumours of minor salivary glands. Cases of pleomorphic adenoma (PA), basal cell adenoma (BCA), canalicular adenoma (CA), adenoid cystic carcinoma (ACC), polymorphous adenocarcinoma (PAC), mucoepidermoid carcinoma (MEC) and epithelial-myoepithelial carcinoma (EMC) were submitted to immunohistochemistry for EMA and DOG1. In PA and BCA, EMA and DOG1 were observed in luminal cells, while in CA the tumour cells were negative for both proteins. The EMA and DOG1 pattern expression detected in EMC was similar to that one observed in benign tumours. In ACC, both myoepithelial e epithelial expressed EMA and DOG-1. PAC tumour cells were only positive for DOG1, whereas MEC were only positive for EMA. In conclusion, EMA and DOG1 expression in benign salivary gland tumours was similar to normal salivary gland tissue and can be used as good marker of tumoral cells derived from intercalated ducts or its progenitor cells, while in malignant salivary gland tumours EMA expression is, however, better used as an indicator of aggressive behavior than a marker of luminal cells.

Benign odontogenic ghost cell lesions revisited and new considerations on dysplastic dentin.

OBJECTIVES: This study aimed to revisit benign odontogenic ghost cell lesions (BOGCL) by hematoxylin and eosin staining and immunohistochemistry. MATERIALS AND METHODS: Thirty cases of calcifying odontogenic cyst (COC) and 6 cases of dentinogenic ghost cell tumor (DGCT) were selected for histopathological and immunohistochemical analysis. Sections stained for cytokeratin (K) 14, K-19, amelogenin, collagen type 1 (COL-1), and dentin matrix acidic phosphoprotein 1 (DMP-1) were evaluated using qualitative analysis. Sections stained for Ki-67 and minichromosome maintenance protein-2 (MCM-2) were evaluated using semi-quantitative analysis. RESULTS: A morphologic overlap was noticed in all BOGCL. Moreover, no differences were detected in the expression of K-14 and K-19. The expression of proliferative markers Ki-67 and MCM-2 was similar between cystic and tumor lesions (p > .05). The presence of COL-1 and absence of amelogenin in the so-called dysplastic dentin, associated with its histologic pattern, suggest that this is in fact an enameloid-like tissue. CONCLUSIONS: The dysplastic dentin should be considered an enameloid-like tissue in these lesions. CLINICAL RELEVANCE: The similarity in histology, protein expression, and proliferative marker indices between COC and DGCT suggest that they are a sole entity and likely represent types of the same neoplasia.

Microvessel density and cell proliferation in juvenile ossifying fibroma: A comparative study with central ossifying fibroma.

Considered as an aggressive counterpart of central ossifying fibroma (OF), juvenile ossifying fibroma (JOF) is a benign fibro-osseous neoplasm characterized by an unpredictable destructive behavior, elevated morbidity, mutilating treatment and high potential for local recurrences. The aim of this study is to compare the analysis for cell proliferation and vascular markers between JOF and OF. Cell proliferation index was measured by Ki-67 and Mcm-2 expression and microvessel density (MVD) was obtained by the immunoexpression of CD34/CD105. We observed a reduced expression of vascular markers, where MVD for CD34 was significantly higher in JOF than in OF (p = 0.009), but no statistical difference was found for CD105. JOF and OF showed low expression for Ki-67 and Mcm-2 and no difference was noted between both, suggesting that other mechanisms such as anti-apoptotic and/or pro-autophagic pathways or even increased expression of matrix metalloproteinases may be responsible for the aggressiveness of JOF.

15d-PGJ2 as an endoplasmic reticulum stress manipulator in multiple myeloma in vitro and in vivo.

Multiple myeloma (MM) is characterised by intense protein folding and, consequently endoplasmic reticulum (ER) stress. The prostaglandin 15d-PGJ2 is able to raise oxidative stress levels within the cell and potentially trigger cell death. The aim of this study was to evaluate the antineoplastic effect of 15d-PGJ2 on MM in vitro and in vivo via ER and oxidative stress pathways. MM.1R and MM.1S cell lines were treated with 15d-PGJ2 at 1-10µM and evaluated with regard to proliferation, mRNA expression of PRDX1, PRDX4, GRP78, GRP94, CHOP, BCL-2 and BAX. Stress data was validated via oxidized glutathione assays. MM.1R cells were inoculated into NOD/SCID mice, which were subsequently treated daily with 15d-PGJ2 at 4mg/kg or vehicle (control), with tumour volume being monitored for 14days. 15d-PGJ2 reduced cell proliferation, induced cell death and apoptosis at 5µM and 10µM and Stress-related genes were upregulated at the same doses. Oxidized glutathione levels were also increased. 15d-PGJ2 at 4mg/kg in vivo halted tumour growth. In conclusion, 15d-PGJ2 induced myeloma cell death via ER stress in vitro. 15d-PGJ2 in vivo also inhibited tumour growth.

Mammaglobin and DOG-1 expression in polymorphous low-grade adenocarcinoma: an appraisal of its origin and morphology.

BACKGROUND: Polymorphous low-grade adenocarcinoma (PLGA) remains a diagnostic challenge for most pathologists due to its large spectrum of histological patterns. In this study, the expression of two new markers recently described for salivary gland tumors was studied in PLGA. METHODS: The morphology of 33 cases of PLGA was carefully evaluated using hematoxylin-and-eosin-stained sections and confirmed by immunohistochemistry for cytokeratin 7, vimentin, and S-100. Periodic acid-Schiff with diastase digestion was also used. The expression of mammaglobin and DOG-1 was carried out using the EnVision System. Mammaglobin was assessed according to the percentage of positively stained tumor cells, while DOG-1 was evaluated according to its presence and site. For MCM-2 and Ki-67, markers of proliferation, the labeling index of cell nuclei positivity was evaluated using total cell number. The ETV6-NTRK3 fusion was examined by fluorescence in situ hybridization analysis. RESULTS: The histological patterns of the tumor were classified as lobular or non-lobular. For the non-lobular pattern, tubular, cribriform, glomeruliform, trabecular, and papillary patterns were observed. Mammaglobin was present in all PLGA cases, and its expression was stronger (P = 0.01) in the lobular than in the non-lobular pattern. The expression of DOG-1 was present in the apical portion and cytoplasm of the cells. Proliferation markers were low for all cases independent of histological pattern. CONCLUSIONS: Polymorphous low-grade adenocarcinoma has been confirmed to originate from the intercalated duct and to feature high expression of mammaglobin in its lobular pattern resembling that of mammary secretory analogue carcinoma, except for the ETV6 gene rearrangement.

Immunoexpression of growth factors and receptors in polymorphous low-grade adenocarcinoma.

BACKGROUND: Polymorphous low-grade adenocarcinoma (PLGA) is a rare malignant tumor that usually arises in the minor salivary glands. Growth factors are cell-secreted peptides that regulate biological processes such as growth, proliferation, and differentiation. In salivary gland tumors, immunoexpression of growth factors and their receptors is associated with cell proliferation, malignant transformation, and tumor invasion. This study analyzed the expression of growth factors and receptors in PLGA, in other to better understand the mechanisms involved in the process of neoplastic cell proliferation and tumor progression. METHODS: The expression of growth factors FGF-2, PDGF-A, PDGF-B and receptors FGFR-1, FGFR-2, PDGFR-α, and EGFR was analyzed in 24 PLGA samples in comparison with normal salivary glands, by immunohistochemistry. A semi-quantitative analysis determined cell positivity in all stained sections. Scores were assigned according to percentage of reactive cells: score 0 < 10%; score 1-10 to 25%; score 2-25% to 50%; score 3->50%. The level of significance was set at 5%. RESULTS: Most of the growth factors and receptors, apart from FGFR-2, were significantly reactive in PLGA. Comparing to salivary acini, all of the reactive growth factors and receptors were significantly stronger in PLGA. Comparing to salivary ducts, the expression of FGF-2, PDGF-B, FGFR-1, and EGFR was significantly stronger in the nuclei and/or cytoplasm of the neoplastic cells. CONCLUSIONS: The increased expression of the growth factors and receptors in the PLGA, compared to normal salivary glands, may be related to cell proliferation, somehow participating in the oncogenic process.

The respiratory research agenda in primary care in Portugal: a Delphi study.

BACKGROUND: A research agenda can help to stimulate and guide research. The International Primary Care Respiratory Group (IPCRG) published a Research Needs Statement (RNS) in 2010 in which 145 research questions were identified. In 2012, priorities for respiratory research were established, based on these questions. To date, there has been no statement on primary care respiratory research needs in Portugal. The aim of the study was to develop a national consensus on research priorities in respiratory diseases in primary care in Portugal and to assess the applicability of the priorities for respiratory research set by the IPCRG. METHOD: We conducted a Delphi study by electronic mail with a panel of experts on respiratory disease from primary and secondary care in Portugal. In the first round, the research needs in respiratory disease in Portugal were identified. In the second round, 196 research questions in six disease areas, derived from the first round and from the IPCRG Respiratory needs statement, were prioritised on a five-point Likert-type scale. In the third round, the questions were prioritized again with feed-back provided on the median scores for each item in the second round. Consensus was considered to have been reached when 80 % of the participants gave a score of 4 or 5 out of five on a given item. RESULTS: The 40 experts identified 121 respiratory research questions in Round 1 and expressed their views on 196 questions in Rounds 2 and 3. Twelve research questions (6 %) reached consensus. There were five questions in the asthma domain on early diagnosis, pulmonary function tests, the use of inhalers, and adherence to treatment. There were four questions in the chronic obstructive pulmonary disease domain on vaccinations, on routine monitoring and evaluation of treatment, on diagnosis, and on adherence to treatments. There was one question in the smoking domain on the effects of brief counselling. There were two questions on respiratory tract infections on the treatment of children and on the prescription of antibiotics. An additional 23 research questions (12 %) achieved consensus between 75 and 79 %. CONCLUSION: The results reflect the Portuguese reality in response the international agenda for research on respiratory diseases published by the IPCRG. They can support the development of future respiratory disease research in Portugal.

Evaluation of Inflammatory Response to Endodontic Sealers in a Bone Defect Animal Model.

AIM: The aim of this study was to evaluate the inflammatory response to MTA Fillapex, AH Plus, and Pulp Canal Sealer Extensive Work Time (EWT), in a murine bone defect grafting model. MATERIALS AND METHODS: Bilateral mandibular critical defects were produced in 45 Wistar rats with a trephine bur#2 and filled with the endodontic sealers. After 7, 14, and 28 days, the rats were euthanized and their jaws were histologically prepared. RESULTS: For the 7-day group, no statistical significance was observed among all studied groups (p > 0.05), and high levels of inflammatory infiltrate were detected. After 14 and 28 days, Pulp Canal Sealer EWT showed statistically lower inflammatory response in comparison to other sealers (p < 0.05) except for the control group (no sealers). CONCLUSION: Pulp Canal Sealer EWT presented the lowest levels of inflammatory response. The critical defect grafting model was an effective method to detect differences among differences on the biological response to endodontic sealers. CLINICAL SIGNIFICANCE: Knowing the biocompatibility of endodontics sealers that will be used in filling the root canal.

The role of FGF-2/HGF and fibronectin matrix on pleomorphic adenoma myoepithelial cell morphology and immunophenotype: an in vitro study.

Myoepithelial cells play a central role in glandular tumors, regulating the progression of in situ to invasive neoplasias, with the tumor microenvironment being shown to be involved in both initiation and progression. This study aimed to analyze the in vitro effects of fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor (HGF) in myoepithelial cells under the influence of the fibronectin matrix extracellular protein. Benign myoepithelial cells were obtained from pleomorphic adenoma and cultured on a fibronectin substratum. FGF-2 and HGF were supplemented at different concentrations and time intervals, in order to evaluate cell proliferation, morphology and immunophenotype. Individually, FGF-2 and HGF supplementation did not alter myoepithelial cell proliferation, morphology or immunophenotype. The fibronectin substratum provoked an increase in cell proliferation and immunopositivity for α-smooth muscle actin and FGF-2. The myoepithelial cell morphology changed when the fibronectin substratum and FGF-2 acted together, highlighting the importance of the fibronectin extracellular matrix protein on the behavior of these cells.

The EGF signaling pathway influences cell migration and the secretion of metalloproteinases by myoepithelial cells in pleomorphic adenoma.

During tumor development, benign neoplastic cells are influenced by the expression of cytokines, growth factors, and proteases present in the tumor microenvironment. Epidermal growth factor (EGF) is the most studied growth factor and is considered important for cell proliferation and migration. Metalloproteinases (MMPs) are also involved in tumor progression. The present study aimed to analyze the proliferation, viability and migration index of pleomorphic adenoma myoepithelial cells, in addition to the secretion of MMPs with EGF supplementation. Benign myoepithelial cells were cultured with two different EGF doses (5 and 10 ng/ml), and the influence of EGF on cell proliferation and viability, using trypan blue and MTT assays, respectively, after 24, 48, and 72 h, was evaluated. To analyze cellular morphology, hematoxylin-eosin staining and indirect immunofluorescence using the anti-vimentin antibody, was performed. In vitro migration assays were performed in Transwell chambers with an 8-µm pore covered with Matrigel and supplemented with 5 or 10 ng/ml of EGF, after 96 h. After 4 days of cell culture, ELISA was performed to determine the MMP-2 and MMP-13 levels. One-way analysis of variance (ANOVA) with post hoc Tukey test was applied, with a significance level of 0.05. The results revealed that EGF influences myoepithelial cell morphology, without alteration of proliferation and viability. The migration assay showed that EGF increased the mean index from 16 % in the control group to 40 and 76 % for 5 and 10 ng/ml of EGF, respectively. ELISA revealed that when the cells were supplemented with either of the EGF doses, an increase in MMP-2 levels was observed when compared with the control group (C). This study concludes that EGF aids in the production of MMP-2, which favors the dissolution of the basement membrane, contributing to cell migration and tumor progression, hence permitting contact between the myoepithelial cells and stroma.

Tooth alterations in areas of bisphosphonate-induced osteonecrosis.

OBJECTIVE: Osteonecrosis of the jaw is a potential side effect when using bisphosphonates. Most studies on the effects of bisphosphonates on teeth have been conducted in vitro or in animal models of tooth development. Therefore, the aim of this study was to describe alterations found in human teeth extracted from areas of bisphosphonate-induced osteonecrosis. MATERIALS AND METHODS: Using a retrospective study design, 16 teeth from 13 patients were extracted from areas of bisphosphonate-induced osteonecrosis during surgical debridement. The specimens were decalcified and embedded in paraffin. A series of 5-µm sections were prepared, stained with hematoxylin and eosin (H&E) and observed under a light microscope. RESULTS: The majority of the patients were female (53.85 %), with a mean age of 60.23 ± 13.18 years. Zoledronate (IV) was the most common bisphosphonate used (92.3 %), over a mean period of 2 years. The commonest alteration observed was hypercementosis (87.5 %), followed by pulpar necrosis (81.25 %), pulp stones attached to the dentine and loose pulp stones in the pulp chamber and root canals in addition to linear calcifications (68.75 %), dentinoid/osteoid material formation (18.75 %), and dental ankylosis (6.25 %). CONCLUSIONS: Patients undergoing bisphosphonate therapy present diverse tooth alterations, which should be closely monitored by clinicians to prevent complications. CLINICAL RELEVANCE: It is paramount that the teeth involved in oral lesions are always examined. Attention should be drawn to the need to establish preventive measures, in terms of dental treatment, for patients prior to starting bisphosphonate therapy.