Specific infiltration of langerin-positive dendritic cells in EBV-infected tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma

We report here the existence of a novel subset of langerin (CD207)-positive, immature dendritic cells (DCs) (CD83(neg)) abundantly infiltrating Epstein Barr virus (EBV)-infected areas in tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma. These CD207(+) DCs differ from conventional epidermal Langerhans cells in their lack of CD1a and CCR6 and their unusual tissue localization. CD207(+) DC infiltration strongly correlates with EBV infection because it was neither detected in EBV negative specimens nor in tissues infected with other human viruses. These immature DCs might represent good candidates for induction of the EBV-specific immune response.

Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment

Oral squamous cell carcinoma (OSCC) is a cancerous lesion with high incidence worldwide. The immunoregulatory events leading to OSCC persistence remain to be elucidated. Our hypothesis is that regulatory T cells (Tregs) are important to obstruct antitumor immune responses in patients with OSCC. In the present study, we investigated the frequency, phenotype, and activity of Tregs from blood and lesions of patients with OSCC. Our data showed that > 80% of CD4(+)CD25(+) T cells isolated from PBMC and tumor sites express FoxP3. Also, these cells express surface Treg markers, such as GITR, CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Purified CD4(+)CD25(+) T cells exhibited stronger suppressive activity inhibiting allogeneic T-cell proliferation and IFN-gamma production when compared with CD4(+)CD25(+) T cells isolated from healthy individuals. Interestingly, approximately 25% of CD4(+)CD25(-) T cells of PBMC from patients also expressed FoxP3 and, although these cells weakly suppress allogeneic T cells proliferative response, they inhibited IFN-gamma and induced IL-10 and TGF-beta secretion in these co-cultures. Thus, our data show that Treg cells are present in OSCC lesions and PBMC, and these cells appear to suppress immune responses both systemically and in the tumor microenvironment.

Tooth Replantation After Use of Euro-Coffins Solution or Bovine Milk as Storage Medium: A Histomorphometric Analysis in Dogs

Purpose: Euro-Collins solution was developed for the preservation of organs for transplantation, whose characteristics have raised interest for its use as a storage medium for avulsed teeth before replantation. This study evaluated histologically and morphometrically the healing process of dog teeth replanted after storage in Euro-Collins solution or bovine milk. Materials and Methods: Eighty roots of 4 young adult mongrel clogs were randomly assigned to 4 groups (n = 20) and the root canals were instrumented and obturated with gutta-percha and a calcium hydroxide-based sealer. After 2 weeks, the teeth were extracted and subjected to the following protocols: GI (negative control), replantation immediately after extraction; GII (positive control), bench-drying for 2 hours before replantation; GIII and GIV, immersion in 10 mL of whole bovine milk and Euro-Collins solution at 4 C, respectively, for 8 hours before replantation. The animals were sacrificed 90 days postoperatively. The pieces containing the replanted teeth were subjected to routine processing for histologic and histometric analyses under light microscopy and polarized light microscopy. Results: Root resorption was observed in all groups. GII exhibited the greatest loss of dental structure (P < .01), and inflammatory resorption was predominant in this group. Storage in milk showed poorer results than immediate replantation and storage in Euro-Collins solution (P < .01). The teeth stored in Euro-Collins solution presented similar extension of root resorption and periodontal ligament reorganization to those of immediately replanted teeth. Conclusions: The findings of this study suggest that the Euro-Collins solution is an adequate storage medium for keeping avulsed teeth for up to 8 hours before replantation. Crown Copyright (C) 2010 Published by Elsevier Inc on behalf of American Association of Oral and Maxillofacial Surgeons. All rights reserved. Oral Maxillofac Surg 68:111-119, 2010

Characterization of CD4(+)CD25(+) natural regulatory T cells in the inflammatory infiltrate of human chronic periodontitis

Periodontitis is an infectious disease, where putative periodontopathogens trigger chronic inflammatory and immune responses against periodontal structures, in which an unbalanced host response is also determinant to the disease outcome. It is reasonable to assume that patient susceptibility to periodontal tissue destruction could be determined by the balance between the response against periodontopathogens and regulatory mechanisms of these events mediated by suppressive T cells. In the present study, we identified and characterized natural regulatory T cells ( Tregs) in the inflammatory infiltrate of human chronic periodontitis ( CP) with emphasis on phenotypic analyses that were carried out to address the participation of Tregs in CP. Results showed that patients with CP presented increased frequency of T lymphocytes and CD4(+)CD25(+) T cells in the inflammatory infiltrate of gingival tissues. These cells exhibited the phenotypic markers of Tregs such as forkhead box p3 ( Foxp3), CTLA- 4, glucocorticoidinducible TNFR, CD103, and CD45RO and seemed to be attracted to the inflammation site by the chemokines CCL17 and CCL22, as their expression and its receptor CCR4 were increased in CP patients. Moreover, besides the increased detection of Foxp3 mRNA, diseased tissues presented high expression of the regulatory cytokines IL-10 and TGF-beta. In addition, the inflammatory infiltrate in CP biopsies was composed of CD25(+)Foxp3(+) and CD25(+)TGF-beta(+) cells, thus corroborating the hypothesis of the involvement of Tregs in the pathogenesis of CP. Finally, these results indicate that Tregs are found in the chronic lesions and must be involved in the modulation of local immune response in CP patients.

The influence of cone-beam computed tomography and periapical radiographic evaluation on the assessment of periapical bone destruction in dog`s teeth

Objective. The aim of this study was to determine the influence of periapical radiographs, cone beam computed tomography (CBCT) sections, and cone beam volumetric data on the determination of periapical bone destruction in endodontically treated distal root canals of premolar canine teeth. Nontreated mesial roots were used as controls. Study design. Enterococcus faecalis strain (ATCC 29212) was inoculated into 30 root canals of 2 mongrel dogs to induce apical periodontitis. After 60 days, the root canals of the distal roots of the 11 mandibular and 4 maxillary premolars were endodontically treated (n = 15). The mesial root canals were used as controls (no treatment). The bone destruction was evaluated after 6 months by 5 evaluators using periapical radiographs and by CBCT (coronal and sagittal sections). After the experimental period, the area of the lesions in periapical radiographs and CBCT sections were measured in mm(2) using the ImageTool software. A single evaluator measured the volumetric data using the OsiriX software. The comparison between the diagnosis methods in treated root canals and controls was performed using parametric and nonparametric criteria. The Pearson correlation coefficient was computed between radiographic values and CBCT volumetric data in treated root canals and controls. Results. The results showed the presence of chronic apical periodontitis in every inoculated tooth. After 6 months, periapical radiographs, coronal CBCT sections, and volumetric data showed lower bone destruction in endodontically treated teeth in comparison with the control group (P < .05). The 5 evaluators found no differences between the apical periodontitis area of treated teeth and controls when CBCT sagittal sections were used (P > .05). No correlation was found between x-ray and CBCT volumetric values in treated root canals. Conclusions. Although selected CBCT sagittal sections showed similar values of bone destruction in endodontically and nontreated root canals, volumetric CBCT data showed that periapical lesions of endodontically treated root canals had half of the volume of periapical lesions in nontreated root canals. No relationship could be found between the periapical values of bone destruction and volumetric data found in CBCT of treated rood canals. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112: 272-279)

Biocompatibility evaluation of alendronate paste in rat`s subcutaneous tissue

Alendronate is a known inhibitor of root resorption and the development of alendronate paste would enhance its utilization as intracanal medication. Therefore, this study aimed to investigate the biocompatibility of experimental alendronate paste in subcutaneous tissue of rats, for utilization in teeth susceptible to root resorption. The study was conducted on 15 male rats, weighing similar to 180-200 grams. The rats` dorsal regions were submitted to one incision on the median region and, laterally to the incision, the subcutaneous tissue was raised and gently dissected for introduction of two tubes, in each rat. The tubes were sealed at one end with gutta-percha and taken as control. The tubes were filled with experimental alendronate paste. The animals were killed at 7, 15 and 45 days after surgery and the specimens were processed in laboratory. The histological sections were stained with hematoxylin-eosin and analyzed by light microscopy. Scores were assigned to the in. ammatory process and statistically compared by the Tukey test (P < 0.05). Alendronate paste promoted severe inflammation process at 7 days, with statistically significant difference compared to the control (P < 0.05%). However, at 15 days, there was a regression of in. ammation and the presence of connective tissue with collagen fibers, fibroblasts and blood vessels was observed. After 45 days, it was observed the presence of well-organized connective tissue, with collagen fibers and fibroblasts, and few in. ammatory cells. No statistical difference was observed between the control and experimental paste at 15 and 45 days. The experimental alendronate paste was considered biocompatible with subcutaneous tissue of rat.

Quantitative analysis of Langerhans` cells in oral chronic graft-vs.-host disease

The Langerhans cells (LCs) are scattered throughout the epithelium of skin and mucosa and have been associated with the graft-vs.-host disease (GVHD), which is the highest cause of morbidity and mortality in patients who underwent bone marrow transplant (BMT). This study aims at quantifying the LCs in the oral chronic GVHD (cGVHD). Microscopic sections from biopsies carried out in the buccal mucosa of 40 patients who underwent allogenic BMT and developed (20) or not (20) oral cGVHD (Groups 1 and 2, respectively) were utilised. For the control group, free surgical margins of 20 biopsies of non-inflammatory lesions in the buccal mucosa (Group 3) were used. The sections were studied in routine colouration and immunostained for CD1a. Group 1 (with cGVHD) presented a greater number of Langerhans` cells/mm(2) (50.6 +/- 37.2) when compared with the other groups (Group 2, 23.11 +/- 19.7; Group 3, 16.6 +/- 17.3). Our results suggest a greater recruitment of LCs in patients transplanted with cGVHD, probably as a result of cytokines secreted by the inflammatory cells.

Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells

Background: Periodontal wound healing and regeneration require that new matrix be synthesized, creating an environment into which cells can migrate. One agent which has been described as promoting periodontal regeneration is an enamel matrix protein derivative (EMD). Since no specific growth factors have been identified in EMD preparations, it is postulated that EMD acts as a matrix enhancement factor. This study was designed to investigate the effect of EMD in vitro on matrix synthesis by cultured periodontal fibroblasts. Methods: The matrix response of the cells was evaluated by determination of the total proteoglycan synthesis, glycosaminoglycan profile, and hyaluronan synthesis by the uptake of radiolabeled precursors. The response of the individual proteoglycans, versican, decorin, and biglycan were examined at the mRNA level by Northern blot analysis. Hyaluronan synthesis was probed by identifying the isotypes of hyaluronan synthase (HAS) expressed in periodontal fibroblasts as HAS-2 and HAS-3 and the effect of EMD on the levels of mRNA for each enzyme was monitored by reverse transcription polymerase chain reaction (RTPCR). Comparisons were made between gingival fibroblast (GF) cells and periodontal ligament (PDLF) cells. Results: EMD was found to significantly affect the synthesis of the mRNAs for the matrix proteoglycans versican, biglycan, and decorin, producing a response similar to, but potentially greater than, mitogenic cytokines. EMD also stimulated hyaluronan synthesis in both GF and PDLF cells. Although mRNA for HAS-2 was elevated in GF after exposure to EMD, the PDLF did not show a similar response. Therefore, the point at which the stimulation of hyaluronan becomes effective may not be at the level of stimulation of the mRNA for hyaluronan synthase, but, rather, at a later point in the pathway of regulation of hyaluronan synthesis. In all cases, GF cells appeared to be more responsive to EMD than PDLF cells in vitro. Conclusions: EMD has the potential to significantly modulate matrix synthesis in a manner consistent with early regenerative events.

Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-1 beta, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1 beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TCF-beta1 and IL-1 beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TCF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the nation of distinct functions for these cell-surface proteoglycans. (C) 2001 Wiley-Liss, inc.

A fluorometric microassay for histamine release from human gingival mast cells

Mast cells are important effector cells of the immune system. We describe a rapid and inexpensive microassay to determine histamine release from human gingival mast cells. The assay is based on the coupling of histamine with o-phthalaldehyde (OPT) at a highly alkaline pH to form a fluorescent product. Using this assay with a sample volume of 10 mul/well in a 384 black well microplate, the histamine detection limit was 0.031 mug/ml. The human mast cell line (HMC-1) and fresh mast cells isolated from human gingival tissue (n = 10) were stimulated with substance P, anti-IgE or calcium ionophore A23187, Calcium ionophore significantly increased histamine release from HMC-1 cells and gingival mast cells (p < 0.05). This microassay will facilitate the study of mast cell histamine release in diseased oral mucosa.

Cytotoxic Effects of Different Concentrations of a Carbamide Peroxide Bleaching Gel on Odontoblast-Like Cells MDPC-23

This study evaluated the cytotoxic effects of a carbamide peroxide (CP) bleaching gel at different concentrations on odontoblast-like cells. Immortalized cells of the MDPC-23 cell line (30,000 cells/cm(2)) were incubated for 48 h. The bleaching gel was diluted in DMEM culture medium originating extracts with different CP concentrations. The amount (mu g/mL) of hydrogen peroxide (H(2)O(2)) released from each extract was measured by the leukocrystal violet/horseradish peroxidase enzyme assay. Five groups (n = 10) were formed according to the CP concentration in the extracts: G1-DMEM (control); G2-0.0001 % CP (0.025 mu g/mL H(2)O(2)); G3-0.001% CP (0.43 mu g/mL H(2)O(2)); G4-0.01% CP (2.21 mu g/mL H(2)O(2)); and G5-0.1 % CP (29.74 mu g/mL H(2)O(2)). MDPC-23 cells were exposed to the bleaching gel extracts for 60 min and cell metabolism was evaluated by the NITT assay. Data were analyzed statistically by one-way ANOVA and Tukey`s test (alpha = 0.05). Cell morphology was examined by scanning electron microscopy. The percentages of viable cells were as follows: G1, 100%; G2, 89.41%; G3, 82.4%; G4, 61.5%; and G5, 23.0%. G2 and G3 did not differ significantly (p > 0.05) from G1. The most severe cytotoxic effects were observed in G3 and G4. In conclusion, even at low concentrations, the CP gel extracts presented cytotoxic effects. This cytotoxicity was dose-dependent, and the 0.1% CP concentration caused the most intense cytopathic effects to the MDPC-23 cells. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 9013: 907-912, 2009

MMP-9 and CD68(+) cells are required for tissue remodeling in response to natural hydroxyapatite

Large bone defects represent major clinical problems in the practice of reconstructive orthopedic and craniofacial surgery. The aim of this study was to examine, through immunohistochemistry approach, the involvement of MMP-9 and CD68(+) cells during tissue remodeling in response to natural hydroxyapatite (HA) implanted in rat subcutaneous tissue. Before experimentation, forty animals were randomly distributed into two experimental groups: Group-I (Gen-Ox (TM) micro-granules) and Group-II (Gen-Ox (TM) macro-granules). Afterwards, the biopsies were collected after 10, 20, 30, and 60 days post-implantation. Our results showed that at 10 days, a low-renewal foreign body type granuloma formation was observed in most of the cases. Macrophage- and fibroblast-like cells were the predominant type of cells positively stained for MMP-9 in both groups. Once macrophage-like cells seemed to be the major source of MMP9, antibody against pan-CD68 epitope was used to correlate these findings. In agreement, MMP-9 and CD68(+) cells were distributed at the periphery and the central region of the granuloma in all experimental periods, however no staining was observed in cell contacting to material. Besides macrophages, the lysosomal glycoprotein epitope recognized by CD68 antibodies can be expressed by mast cell granules and sometimes by fibroblasts. Taken together, our results suggest that xenogenic HA promotes extracellular matrix remodeling through induction of MMP-9 activity and presence of CD68(+) cells.

Characterization of a Local Renin-Angiotensin System in Rat Gingival Tissue

Background: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. Methods: Reverse transcription - polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). Results: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang 1 (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. Conclusion: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro. J Periodontol 2009;80:130-139.

gamma-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of gamma-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca(2+) handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca(2+), reduced amount of intrareticular Ca(2+), and reduced capacitive Ca(2+) entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FIVIK) during the 3 day period after irradiation, and by the chelator of intracellular Ca(2+), 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca(2+), amount of intrareticular Ca(2+), capacitative Ca(2+) entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca(2+) handling, and apoptosis appear due to a toxic action of intracellular Ca(2+). Ca(2+)-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca(2+) handling and apoptosis induced by gamma-radiation. (c) 2008 Elsevier B.V. All rights reserved.

Osteointegration of Autogenous Bone Graft Associated With Osteoblastic Cells Under Treatment With Caffeine

Purpose: The present study investigated osteointegration of autogenous bone (AB) from calvaria graft associated with osteoblastic cells (OC) in bone defects in rats subjected to daily administration of caffeine. Materials and Methods: Male rats received daily intraperitoneal injection of 1.5% caffeine (0.2 mL/100 g body weight) or saline solution for 30 days. Then they were anesthetized, submitted to the extraction of the upper right incisor, and implanted with AB only and AB + OC. The animals were killed on 7th, 21st, and 42nd days after surgery, and their maxilla were processed for obtaining semiserial sections (5 mu m) stained with hematoxylin and eosin. Through image analysis system, the bone volume and the quality of graft in adjacent areas were estimated. Results: The results showed that in caffeine treatment, the AB + OC graft showed no foreign body and acute inflammatory reactions inside the defect when compared to AB. The histometric results revealed that the association AB + OC produced significant increase (10%-15%) in bone volume in later experimental period (42 days) when compared with saline solution group (P <= 0.01). Conclusions: It was concluded that the association of AB from calvaria + OC demonstrated progressive osteointegration and accelerated the repair of bone defects in animals treated with daily caffeine. (Implant Dent 2011;20:369-373)