Expression of Extracellular Matrix Proteins in Human Dental Pulp Stem Cells Depends on the Donor Tooth Conditions

Introduction: Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. An important source of adult stem cells is the dental pulp. In dentistry, regenerative strategies are of importance because of hard dental tissue damage especially as result of caries lesions, trauma, or iatrogenic procedures. The regeneration of dental tissues relies on the ability of stem cells to produce extracellular (ECM) proteins encountered in the dental pulp tissue. Thus, the aim of this study was to analyze the expression and distribution of proteins encountered in dental pulp ECM (type I collagen, fibronectin, and tenascin) in stem cells. Methods: Human immature dental pulp stem cells (hIDPSCs) from deciduous (DL-1 and DL-4 cell lines) and permanent (DL-2) teeth were used. The distribution of ECM proteins was observed using the immunofluorescence technique. The gene expression profile was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Positive reactions for all ECM proteins were observed independently of the hIDPSCs analyzed. Type I collagen appeared less evident in DL-2 than in other hIDPSCs. Fibronectin and tenascin were less clear in DL-4. The RT-PCR reactions showed that type I collagen was lesser expressed in the DL-2 cells, whereas fibronectin and tenascin were similarly expressed in all hIDPSCs. Conclusions: The distribution and expression of ECM proteins differ among the hIDPSCs. These differences seemed to be related to the donor tooth conditions (deciduous or permanent, retained or erupted, and degree of root reabsorption). (J Endod 2010;36:826-831)

Expression of Homeobox Genes in Oral Squamous Cell Carcinoma Cell Lines Treated With All-Trans Retinoic Acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on clay 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437-1444, 2010. (C) 2010 Wiley-Liss, Inc.

Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma

Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p = 0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p = 0.010 and p = 0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC. (C) 2009 Elsevier Ltd. All rights reserved.

Glial fibrillary acidic protein in tumor types with cartilaginous differentiation

Glial fibrillary acidic protein (GFAP) is a member of the intermediary filament protein family. It is an important component of astrocytes and a known diagnostic marker of glial differentiation. GFAP is expressed in other neural tumors and pleomorphic adenoma and, less frequently, in cartilage tumors, chordomas, and soft tissue myoepitheliomas. The aim of this study was to evaluate the role of GFAP and its reliability in nonglial tumors as an immunohistochemical marker. We evaluated GFAP gene and protein expression using Q-PCR and immunohistochemistry, respectively, in 81 and 387 cases of soft tissue, bone tumors, and salivary pleomorphic adenomas. Immunohistochemistry staining for GFAP was observed in all osteosarcomas (8 cases), all pleomorphic adenomas (7 cases), in 5 of 6 soft tissue myoepitheliomas, and in 21 of 76 chondrosarcomas. By Q-PCR, GFAP was highly expressed in pleomorphic adenomas and, to a lesser extent, chondrosarcomas, soft tissue myoepitheliomas, and chondroblastic osteosarcomas. The results that we obtained by immunohistochemistry and Q-PCR were well correlated. GFAP is a potential marker for tumors with cartilaginous differentiation, supported by evidence that GFAP is expressed in certain cases of myoepithelial tumors by immunohistochemistry, including soft tissue myoepitheliomas, which are related to cartilaginous differentiation. These findings contribute significantly to the diagnosis of soft tissue myoepitheliomas with cartilaginous differentiation and chondroblastic osteosarcoma in mesenchymal tumors. Modern Pathology ( 2009) 22, 1321-1327; doi: 10.1038/modpathol.2009.99; published online 7 August 2009

Isolation of Candida dubliniensis from denture wearers

Candida albicans is considered the most important Candida species able to cause oral infections in denture wearers. In recent years, Candida dubliniensis has emerged as a pathogenic yeast in humans. The close phenotypic similarities of C. albicans and C. dubliniensis have led to the misidentification of these species. In this work, our aim was to verify through PCR the presence of C. dubliniensis in palate and maxillary denture samples from 112 denture wearers presenting with or without denture-related stomatitis (DRS). C. dubliniensis was isolated at low rates from both palate (5.3% and 10.7%) and maxillary denture (5.3% and 8.9%) samples from wearers regardless of the presence of the disease. However, when C. dubliniensis was detected in individuals with DRS, it was always associated with C. albicans. In addition, our results showed that C. albicans was the most commonly identified candidal species in maxillary denture and hard palate samples from DRS patients (78.5% and 89.2%, respectively) as well as from controls (31.2% and 28.5%, respectively). In conclusion, C. dubliniensis was detected in the oral environment of denture wearers. The association of C. dubliniensis with C. albicans occurred in approximately 10% of the DRS cases.

In vivo Acid Etching Effect on Bacteria within Caries-Affected Dentin

Acid etching procedures may disrupt residual bacteria and contribute to the success of incomplete caries removal followed by adhesive restoration. This study evaluated the in vivo effect of acid etching on cariogenic bacterial activity within affected dentin after minimally invasive treatment of caries lesions. Twenty-eight carious permanent teeth received standardized selective caries removal and random acid etch treatment (E) or not (NE) prior to adhesive restoration. Baseline and 3-month dentin biopsies were collected. The number of bacteria and activity of total bacterial cells and Streptococcus mutans were determined by quantitative PCR and RT-PCR. No statistically significant differences were observed in total bacterial number and activity between E and NE treatments (p > 0.3008). For NE, however, the residual S. mutans bacterial cells were reduced (p = 0.0027), while the activity per cell was significantly increased (p = 0.0010) after reentry at 3 months after restoration. This effect was not observed in group E. Although no significant differences were found between groups, this study suggests that acid etching of affected dentin prior to adhesive restoration may directly or indirectly have an inhibitive effect on the activity of residual cariogenic bacteria. Further research is required to investigate this potential effect. Copyright (C) 2010 S. Karger AG, Basel

Differential patterns of receptor activator of nuclear factor kappa B ligand/osteoprotegerin expression in human periapical granulomas: Possible association with progressive or stable nature of the lesions

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.

A comparison of the clinical anesthetic efficacy of 4% articaine and 0.5% bupivacaine (both with 1 : 200,000 epinephrine) for lower third molar removal

Objective: This study compared the clinical efficacy of 4% articaine (A200) and 0.5% bupivacaine (B200), both with 1: 200,000 epinephrine, for lower third molar removal. Study design: Fifty patients underwent removal of symmetrically positioned lower third molars, in 2 separate appointments, under local anesthesia either with A200 or B200, in a double-blind, randomized, and crossover manner. Time to onset, duration of postoperative analgesia, duration of anesthetic action on soft tissues, intraoperative bleeding, and hemodynamic parameters were evaluated. Results: A statistically significant difference between the time to onset of A200 (1.66 +/- 0.13 minutes) and B200 (2.51 +/- 0.21 minutes) was found (P < .05). There was no statistically significant difference in the duration of analgesia, whether the patient was subjected to osteotomy or not, regardless of the local anesthetic used (3 to 4 hours; P < .05). However, when patients received B200 they experienced a statistically significant longer period of anesthesia on the soft tissues as compared with when they had received A200 (around 5 hours and 4 hours, respectively, P < .05). The surgeon`s rating of intraoperative bleeding was considered very close to minimal for both anesthetics. In the surgeries with osteotomy, the comparison between A200 and B200 showed statistically significant differences in the diastolic (64 mm Hg and 68 mm Hg, respectively, P = .001) and mean arterial pressure (86 mm Hg and 89 mm Hg, respectively, P = .031) when data from all the surgical phases were pooled. Additionally, the mouth opening at the suture removal was statistically different for A200 and B200 solutions (91.90% +/- 3.00% and 88.57% +/- 2.38% of the preoperative measure, respectively) when surgeries required bone removal (P < .05). Conclusions: In comparison with 0.5% bupivacaine, 4% articaine (both with 1: 200,000 epinephrine) provided a shorter time to onset and comparable hemostasis and postoperative pain control with a shorter duration of soft tissue anesthesia in lower third molar removal.

GABAergic antagonist blocks the reduction of tonic immobility behavior induced by activation of 5-HT(2) receptors in the basolateral nucleus of the amygdala in guinea pigs

Tonic immobility (TI) is a temporary state of profound motor inhibition induced by situations that generate intense fear, with the objective of protecting an animal from attacks by predators. A preliminary study by our group demonstrated that microinjection into the basolateral nucleus of the amygdala (BLA) of an agonist to 5-HT(1A) and 5-HT(2) receptors promoted a decrease in TI duration. In the current study, the effects of GABAergic stimulation of the BLA and the possible interaction between GABA(A) and 5-HT(2) receptors on TI modulation were investigated. Observation revealed that GABAergic agonist muscimol (0.26 nmol) reduced the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol) increased TI duration. Additionally, microinjection of 5-HT(2) agonist receptors (alpha-methyl-5-HT, 0.32 nmol) into the BLA decreased TI duration, an effect reversed by pretreatment with bicuculline (at the dose that had no effect per se, 0.2 nmol). Moreover, the activation of GABA(A) and 5-HT(2) receptors in the BLA did not alter the spontaneous motor activity in the open field test. These experiments demonstrated that the activation of GABA(A) and 5-HT(2) receptors of the BLA possibly produce a reduction in unconditioned fear that decreases the TI duration in guinea pigs, but this is not due to increased spontaneous motor activity, which could affect a TI episode nonspecifically. Furthermore, these results suggest an interaction between GABAergic and serotoninergic mechanisms mediated by GABA(A) and 5-HT(2) receptors. In addition, the GABAergic circuit of the BLA presents a tonic inhibitory influence on TI duration. (C) 2009 Elsevier Inc. All rights reserved.

The Effect of TAK-778 on Gene Expression of Osteoblastic Cells Is Mediated Through Estrogen Receptor

This study evaluated the effect of TAK-778 [(2R, 4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide)] on in vitro osteogenic events and on gene expression of osteoblastic cells derived from human alveolar bone and the participation of estrogen receptors (ERs) on such effect. Osteoblastic cells were subcultured, with or without TAK-778 (10(-5) M), to evaluate cell growth and viability, total protein content, and alkaline phosphatase (ALP) activity at 7, 14, and 21 days; bone-like formation at 21 days; and gene expression, using cDNA microarray, at 7 days. Also, osteoblastic cells were exposed to TAK-778 (10-5 M) combined to ICI182,780, a nonspecific ER antagonist (10(-6) M), and gene expression was evaluated by real-time polymerase chain reaction (PCR) at 7 days. TAK-778 induced a reduction in culture growth and an increase in cell synthesis, ALP activity, and bone-like formation. The cDNA microarray showed genes associated with cell adhesion and differentiation, skeletal development, ossification, and transforming growth factor-P receptor signaling pathway, with a tendency to be higher expressed in cells exposed to TAK-778. The gene expression of ALP, osteocalcin, Msh homeobox 2, receptor activator of NF-kappa B ligand, and intercellular adhesion molecule 1 was increased by TAK-778 as demonstrated by real-time PCR, and this effect was antagonized by ICI182,780. The present results demonstrated that TAK-778 acts at a transcriptional level to enhance the in vitro osteogenic process and that its effect on gene expression of osteoblastic cells is mediated, at least partially, through ERs. Based on these findings, TAK-778 could be considered in the treatment of bone metabolic disorders. Exp Biol Med 234:190-199, 2009

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress. we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201 +/- 5 vs. 163 +/- 7 mm Hg in diabetic rats untreated and treated with lercaniclipine, respectively; P < 0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P < 0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P < 0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P < 0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2MMP-2 expression. (C) 2008 Published by Elsevier B.V.

5-HT1A, but not 5-HT2 and 5-HT7, receptors in the nucleus raphe magnus modulate hypoxia-induced hyperpnoea

Aim: In the present study, we assessed the role of 5-hydroxytryptamine (5-HT) receptors (5-HT1A, 5-HT2 and 5-HT7) in the nucleus raphe magnus (NRM) on the ventilatory and thermoregulatory responses to hypoxia. Methods: To this end, pulmonary ventilation (V-E) and body temperature (T-b) of male Wistar rats were measured in conscious rats, before and after a 0.1 mu L microinjection of WAY-100635 (5-HT1A receptor antagonist, 3 mu g 0.1 mu L-1, 56 mM), ketanserin (5-HT2 receptor antagonist, 2 mu g 0.1 mu L-1, 36 mM) and SB269970 (5-HT7 receptor antagonist, 4 mu g 0.1 mu L-1, 103 mM) into the NRM, followed by 60 min of severe hypoxia exposure (7% O-2). Results: Intra-NMR microinjection of vehicle (control rats) or 5-HT antagonists did not affect V-E or T-b during normoxic conditions. Exposure of rats to 7% O-2 evoked a typical hypoxia-induced anapyrexia after vehicle microinjections, which was not affected by microinjection of WAY-100635, SB269970 or ketanserin. The hypoxia-induced hyperpnoea was not affected by SB269970 and ketanserin intra-NMR. However, the treatment with WAY-100635 intra-NRM attenuated the hypoxia-induced hyperpnoea. Conclusion: These data suggest that 5-HT acting on 5-HT1A receptors in the NRM increases the hypoxic ventilatory response.