S100 Proteins as Biomarkers in Risk Estimations for Malignant Transformation in Oral Lesions.

Oncologic relevant members of S100 proteins are described as promising biomarkers in molecular pathology for risk estimation in oral neoplasia exhibiting different stages of malignancy: gingiva as healthy tissue, irritation fibroma as benign, leukoplakia as precancerous, and oral squamous cell carcinoma as malignant entity. Gene expression levels of S100A4 (metastasin), S100A7 (psoriasin), S100A8 (calgranulin A), and S100A9 (calgranulin B) were analyzed using quantitative RT-PCR. In addition, immunohistochemistry-based microscopy was used to examine cellular localization and distribution of these biomarkers in tissue sections. The results indicate that S100 proteins represent promising biomarkers for early-stage diagnosis in oral lesions. The inclusion of expression profiles and ratios for each entity even improves their diagnostic validity.

Insulin-like growth factor 1 (IGF1) affects proliferation and differentiation and wound healing processes in an inflammatory environment with p38 controlling early osteoblast differentiation in periodontal ligament cells.

OBJECTIVE: The objective of this study was to investigate effects of insulin-like growth factor 1 (IGF1) on proliferation, wound healing and differentiation processes of human periodontal ligament (PDL) cells under inflammatory conditions and whether the protective, anabolic effects of IGF1 can attenuate unfavorable effects of interleukin-1ß (IL-1ß). DESIGN: Inflammation was mimicked through cell stimulation with IL-1ß. PDL cells were characterized in respect to the presence of components of the IGF system and the responsive potential on IL-1ß incubation. Gene expression levels were analyzed by quantitative real-time PCR. Cellular localization of target proteins was visualized using fluorescent-based immunohistochemistry. Effects on cell division were investigated by proliferation assays. Wound healing was analyzed using light microscopic techniques. Differentiation was quantified by measuring biomineralization and osteoblast-specific alkaline phosphatase enzyme activity. RESULTS: PDL cell proliferation and wound healing were positively affected by IGF1 and the combination of IGF1 with IL-1ß, while only IL-1ß showed negative effects. Biomineralization was enhanced by IGF1, IL-1ß, and the combination of both stimulants. Osteoblast differentiation was increased by IL-1ß and the combination of IL-1ß with IGF1, whereas only IGF1 negatively affected ALP activity. Phosphorylation of p38 was regulated by IL-1ß and IGF1. CONCLUSIONS: The data presented in this work showed a potential of IGF1 to improve wound healing and proliferation processes and to sustain cell differentiation under inflammatory stimuli in PDL cells.

Cellular Distribution and Gene Expression Pattern of Metastasin (S100A4), Calgranulin A (S100A8), and Calgranulin B (S100A9) in Oral Lesions as Markers for Molecular Pathology.

The objective of this study was to analyze cellular localization and expression levels of oncologic relevant members of the S100 family in common oral lesions.Biopsies of various oral lesions were analyzed. S100A4 showed a higher expression rate in leukoplakias and oral squamous cell carcinomas. Transcript levels of S100A8 and S100A9 were significantly decreased in malignant OSCCs. A correlation could be drawn between the expression levels of these genes and the pathological characteristics of the investigated lesions. S100A4, A8, and A9 proteins represent promising marker genes to evaluate the risk potential of suspicious oral lesions in molecular pathology.

Ghrelin promotes oral tumor cell proliferation by modifying GLUT1 expression.

In our study, ghrelin was investigated with respect to its capacity on proliferative effects and molecular correlations on oral tumor cells. The presence of all molecular components of the ghrelin system, i.e., ghrelin and its receptors, was analyzed and could be detected using real-time PCR and immunohistochemistry. To examine cellular effects caused by ghrelin and to clarify downstream-regulatory mechanisms, two different oral tumor cell lines (BHY and HN) were used in cell culture experiments. Stimulation of either cell line with ghrelin led to a significantly increased proliferation. Signal transduction occurred through phosphorylation of GSK-3ß and nuclear translocation of ß-catenin. This effect could be inhibited by blocking protein kinase A. Glucose transporter1 (GLUT1), as an important factor for delivering sufficient amounts of glucose to tumor cells having high requirements for this carbohydrate (Warburg effect) was up-regulated by exogenous and endogenous ghrelin. Silencing intracellular ghrelin concentrations using siRNA led to a significant decreased expression of GLUT1 and proliferation. In conclusion, our study describes the role for the appetite-stimulating peptide hormone ghrelin in oral cancer proliferation under the particular aspect of glucose uptake: (1) tumor cells are a source of ghrelin. (2) Ghrelin affects tumor cell proliferation through autocrine and/or paracrine activity. (3) Ghrelin modulates GLUT1 expression and thus indirectly enhances tumor cell proliferation. These findings are of major relevance, because glucose uptake is assumed to be a promising target for cancer treatment.

Freehand Condyle-Positioning During Orthognathic Surgery: Postoperative Cone-Beam Computed Tomography Shows Only Minor Morphometric Alterations of the Temporomandibular Joint Position.

OBJECTIVES: Purpose of this study was to evaluate changes in the temporomandibular joint (TMJ) position after bilateral sagittal split osteotomy (BSSO) of the mandible by the help of pre- and postoperative cone-beam computed tomography (CBCT) images. MATERIALS AND METHODS: A collective of n = 78 patients was investigated between 2009 and 2011 before and after BSSO of the mandible in mono- or bimaxillary orthognathic surgery procedures. No intraoperative fixation of the condyles was administered. CBCT scans were performed in all patients before and immediately after surgery with the KaVo 3DeXam device in the position of terminal occlusion. Subsequently, all scans were analyzed by help of the eXam Vision program and the ImageJ image processing software. Alterations of the TMJs were quantified by determining pre- to postoperative differences of the intercondylar distance, the mandibular angle on both sides, and the condylar angles in the transversal plane. RESULTS: The difference between pre- and postoperatively ascertained values was minimal (means: lateral condylar distance -0.17  mm; distance of condylar centers -0.32  mm; medial condylar distance -0.49  mm; left mandibular angle +1.06°; right mandibular angle +2.06°; condylar angles in relation to a reference line: left -2.93, right -0.75; angle of cutting +3.42). There is no apparent tendency toward a positional change in any of the 3 examined planes. Between bi- and monomaxillarily operated patients there was no difference either, except for the osteotomy plane. CONCLUSIONS: A 3-dimensional analysis of CBCT data of the TMJ seems to be appropriate to determine the condylar position pre- and postoperatively. Performed by an experienced orthognathic surgeon, BSSO of the mandible does not effectuate any relevant changes of the TMJ-position, thus making an intraoperative condyle-fixation unnecessary.

[Ridge preservation with synthetic nanocrystalline hydroxyapatite reduces the severity of gingival invaginations-a prospective clinical study]. / Alveolenerhalt mit einem nanopartikulärem Hydroxylapatit verringert den Ausprägungsgrad von Gingivaduplikaturen - eine prospektive klinische Studie.

BACKGROUND AND OBJECTIVE: Gingival invaginations develop after tooth extraction and subsequent orthodontic space closure. Aetiological factors and long-term effects of gingival invaginations on oral health are nearly unknown. In addition, preventive or therapeutic strategies are rare. This prospective clinical study employing the split mouth technique was performed to investigate the effect of extraction socket augmentation with a synthetic nanocrystalline hydroxyapatite (NanoBone(®) Artoss, Rostock, Germany) on the incidence and degree of gingival invaginations. MATERIAL AND METHODS: A total of 10 orthodontic patients with need for symmetric premolar extractions offering a total of 28 extractions were included in this trial. The study plan provided one extraction site to be augmented with synthetic nanocrystalline hydroxyapatite (NanoBone(®)), the other served as control. After primary wound healing, space closure was performed under defined biomechanical conditions. After space closure was accomplished, occurrence and degree of gingival invaginations as well as probing depths of the adjacent teeth mesial and distal to the extractions were determined and dental radiographs were taken. RESULTS: The degree of gingival invaginations and probing depths mesial and distal of the extraction were significantly reduced on NanoBone(®) augmented extraction sites. In addition, 70% of the radiographs revealed translucent and hyperdense areas on the intervention side after space closure. Apical root resorption was found in 2 patients on both the NanoBone(®) side and the control side. CONCLUSION: Ridge preservation with NanoBone(®) appeared to reduce the severity of gingival invaginations. Further investigation on long-term effects is mandatory to eliminate the appearance of adverse effects.

Modified triaca-style wing osteotomy to correct facial asymmetry in a patient with horizontal growth-type condylar hyperplasia.

In this short communication, we suggest a slight modification of Albino Triaca's chin-wing osteotomy to vertically correct the inferior border of the mandible in a patient with horizontal growth-type facial asymmetry due to condylar hyperplasia.

Human α-defensin (DEFA) gene expression helps to characterise benign and malignant salivary gland tumours.

BACKGROUND: Because of the infrequence of salivary gland tumours and their complex histopathological diagnosis it is still difficult to exactly predict their clinical course by means of recurrence, malignant progression and metastasis. In order to define new proliferation associated genes, purpose of this study was to investigate the expression of human α-defensins (DEFA) 1/3 and 4 in different tumour entities of the salivary glands with respect to malignancy. METHODS: Tissue of salivary glands (n=10), pleomorphic adenomas (n=10), cystadenolymphomas (n=10), adenocarcinomas (n=10), adenoidcystic carcinomas (n=10), and mucoepidermoid carcinomas (n=10) was obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of DEFA 1/3 and 4 were analyzed by quantitative realtime PCR and compared with healthy salivary gland tissue. Additionally, the proteins encoded by DEFA 1/3 and DEFA 4 were visualized in paraffin-embedded tissue sections by immunohistochemical staining. RESULTS: Human α-defensins are traceable in healthy as well as in pathological altered salivary gland tissue. In comparison with healthy tissue, the gene expression of DEFA 1/3 and 4 was significantly (p<0.05) increased in all tumours - except for a significant decrease of DEFA 4 gene expression in pleomorphic adenomas and a similar transcript level for DEFA 1/3 compared to healthy salivary glands. CONCLUSIONS: A decreased gene expression of DEFA 1/3 and 4 might protect pleomorphic adenomas from malignant transformation into adenocarcinomas. A similar expression pattern of DEFA-1/3 and -4 in cystadenolymphomas and inflamed salivary glands underlines a potential importance of immunological reactions during the formation of Warthin's tumour.

IL-23-producing CD68(+) macrophage-like cells predominate within an IL-17-polarized infiltrate in chronic periodontitis lesions.

AIM: To analyse antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages (Mo) or B cells depending on the regional site of chronic periodontitis (CP), and to investigate their relation to Th17 cells. MATERIAL AND METHODS: Biopsies from oral mucosa as well as the coronal and bottom regions of CP were analysed by immunhistochemistry, immunofluorescence, flow cytometry and real-time PCR. RESULTS: A predominance of CD68(+) Mo-like cells and CD20(+) B cells and strong Th17 infiltration was observed in the bottom region of CP lesions, while CD1a(+) DCs were only detected in the coronal regions, where Th17 infiltration was low. Furthermore, CD68(+) Mo-like cells displayed CD163 expression as a typical Mo-marker, but expressed in parallel typical DCs markers, such as CD11c or CD209 and TLR4. Interestingly, Th17-inducing cytokine IL-23p19 was produced by CD68(+) Mo-like cells, but not CD20(+) B cells. Moreover, the stimulation of in vitro generated CD68(+) Mo-like cells by Porphyromonas gingivalis-derived (Pg) lipopolysaccharide resulted in the upregulation of their IL-23p19 mRNA expression, which was inhibited by the blockage of TLR4. CONCLUSIONS: In view of these data, a picture emerges that IL-17-producing cells in CP could be in part directed by CD68(+) Mo-like cells, which produce IL-23p19 upon TLR4 activation by Pg.

Risk estimation for a malignant transformation of oral lesions by S100A7 and Doc-1 gene expression.

The objective of this study was the correlation of Doc-1- and S100A7-gene expression in common oral lesions with their cancerous-transformation risk. Biopsies (n = 15 each) of healthy gingiva, irritation fibromas, leukoplakias and Oral squamous cell carcinoma (OSCCs) were obtained, and after RNA-extraction, transcripts of Doc-1 and S100A7 were quantified by RT-PCR. In comparison with the healthy gingiva, the expression of Doc-1 was decreased, whereas the expression of S100A7 was upregulated in all lesions. As the extent of Doc-1-inactivation and S100A7-overexpression is correlated with their biological behavior, the combined investigation of both genes could be a promising marker in intraoral lesions to estimate the risk for their malignant transformation.

Human beta-defensin-1, -2, and -3 exhibit opposite effects on oral squamous cell carcinoma cell proliferation.

The objective of this study was to investigate the impact of human beta-defensins (hBDs) on oral squamous cell carcinoma (OSCC) proliferation and hBD expression in vitro. BHY-OSCC cell lines were stimulated with hBD-1, -2, and -3. Proliferation of BHY cells was ascertained and hBD-mRNA expression was evaluated by real-time PCR. Proliferation of BHY cells decreased by 25% in response to hBD-1 stimulation but increased after stimulation with hBD-2 and -3. HBD-1 stimulation enhanced hBD-3 expression, whereas HBD-2 stimulation decreased early hBD-3 expression. HBD-3 stimulation enhanced hBD-1 expression. HBDs profoundly impact on OSCC proliferation and hBD expression in vitro. Therefore, hBD-1 might function as a tumor suppressor gene in OSCCs, while hBD-2 and -3 might be protooncogenes.

High α-defensin and S100A7 expression and missing DOC-1 down-regulation characterize irritation fibromas of the oral cavity and may counteract malignant transformation.

PURPOSE: The purposes of this study were to analyze the gene expression pattern of antimicrobial peptides, tumor suppressors, growth factors, matrix metalloproteases, and inflammatory cytokines and chemokines in oral irritation fibromas and to identify genes with protective effects against malignant transformation in benign proliferating tumors of the oral mucosa. MATERIALS AND METHODS: Biopsies of irritation fibromas (n = 15) and healthy gingiva (n = 15) were obtained during routine surgical procedures. RNA was extracted according to standard protocols, and transcription levels of CCL20, DEFA 1/3, DEFA 4, S100A7, DOC-1, interleukin (IL) 1ß, IL-6, IL-8, IL-10, tumor necrosis factor α, Cox-2, matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, transforming growth factor ß1, transforming growth factor α, and keratinocyte growth factor were analyzed by real-time polymerase chain reaction. In addition, immunostaining was performed to visualize the transcription products of the genes of interest in fibroma tissue as well as in healthy gingiva. RESULTS: The gene expression of S100A7 was 11.3-fold and that of DEFA 1/3 was 14-fold higher in irritation fibromas than in healthy gingiva, whereas the expression of MMP-3 and of inflammation markers IL-1ß, IL-6, IL-8, tumor necrosis factor α, and Cox-2 was reduced. Profound down-regulation of DOC-1 gene expression, characteristic for proliferating malignant tumors of the oral cavity, was in irritation fibromas not verifiable. CONCLUSIONS: Changes in the expression pattern of S100A7, DEFA 1/3, and MMP-3 seem to be involved in the development of irritation fibromas, whereas chronic inflammation might be of less importance. Overexpression of S100A7, but missing down-regulation of the tumor-suppressor gene DOC-1, might exert protective effects and counteract malignant transformation of benign, proliferating lesions of the oral cavity.

Phl p 5 resorption in human oral mucosa leads to dose-dependent and time-dependent allergen binding by oral mucosal Langerhans cells, attenuates their maturation, and enhances their migratory and TGF-beta1 and IL-10-producing properties.

BACKGROUND: Sublingual immunotherapy (SLIT) is safe and effective as treatment of allergic rhinitis and mild asthma. Oral mucosal Langerhans cells (oLCs) play a central role. However, little is known about allergen binding by oLCs during mucosal allergen resorption and its impact on oLC functions. OBJECTIVE: Binding of Phl p 5 to oLCs was studied in a standardized ex vivo model to investigate mechanisms important for SLIT. METHODS: Human oral mucosal biopsies were incubated with the grass pollen allergen Phl p 5. Migration, binding of Phl p 5, phenotype and cytokine production, and T-cell priming of Phl p 5-binding oLCs were analyzed. RESULTS: Significant uptake required more than 5 minutes, and dose-dependent binding of Phl p 5 to oLCs was saturated at 100 microg/mL Phl p 5. Furthermore, Phl p 5 significantly increased the migratory capacity of oLCs but attenuated their maturation and strongly promoted the release of TGF-beta1 and IL-10 by oLCs themselves as well as by cocultured T cells. CONCLUSION: Oral mucosal Langerhans cells bind Phlp5 in a dose-dependent and time-dependent manner, leading to an increased production of tolerogenic cytokines and an enhanced migratory capacity but decelerated maturation of oLCs.

Down regulation and nuclear localization of human beta-defensin-1 in pleomorphic adenomas of salivary glands.

Although antimicrobial peptides (AMPs) appear to have diverse functional activities in innate immunity, a few reports suggest a potential role of human beta-defensin (hBD)-1 in tumor suppression. The aim of the present study was to compare the expression patterns of hBD-1, -2 and -3 in various features of human salivary gland tissues, such as healthy parenchyma, chronic sialadenitis and intraglandular pleomorphic adenomas, with their adjacent normal tissues. Twenty human salivary gland specimens (five healthy, five chronic sialadenitis, five pleomorphic adenomas and five adenoma adjacent normal tissues (AANTs)) were investigated for mRNA expression levels of hBD-1, -2 and -3 by quantitative real-time RT-PCR. Additionally, immunohistochemistry for the hBD-1, -2 and -3 peptides was performed for analysis of localization. Considerably increased, 80-fold higher hBD-1 and increased hBD-3 mRNA expression levels compared to healthy salivary gland tissues were detected in chronic sialadenitis. In pleomorphic adenomas hBD-2 expression levels were lower, but hBD-1 expression levels were significant decreased (p=0.03) compared to healthy parenchyma. Interestingly, the AANTs showed a 48-fold higher expression of hBD-1 and increased hBD-3 expression compared to the healthy salivary gland. Immunohistochemistry of the tumors showed nuclear hBD-1 localization. For the first time, it was shown that hBD-1 gene expression is significantly decreased in pleomorphic adenomas, while simultaneously the protein is localized in the nucleus. Increased expression levels in glandular inflammation have been described previously albeit not in AANTs. These data support the hypothesis that hBD-1 might be a potential tumor suppressor also in benign salivary gland tumors in addition to other genetic alterations.

Hyperplasia of the coronoid process in patients with ankylosing spondylitis (Bechterew disease).

Although involvement of the temporomandibular joint in patients with ankylosing spondylitis (AS, Bechterew disease) has been described previously, hyperplasia of the mandibular coronoid process in those patients has not been reported yet. Case notes were studied, and records were made of age, sex, clinical symptoms, radiography, and treatment in all patients with a confirmed diagnosis of coronoid hyperplasia presenting at the Department of Oral and Maxillofacial Surgery, University of Bonn, between 1995 and 2007. Sixteen cases of coronoid hyperplasia were recruited, of which 12 were bilateral and 4 were unilateral. Four patients had AS, 3 of them were HLA-B27-positive. Temporomandibular joint symptoms are frequently seen in patients with AS. Nevertheless, it must be considered that a limitation of jaw mobility in those patients might also be caused by an elongation of the mandibular coronoid process.

Family-based association study of the MTHFR polymorphism C677T in patients with nonsyndromic cleft lip and palate from central Europe.

OBJECTIVE: The 677C-->T allele in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene has been implicated in the etiology of nonsyndromic cleft lip and palate (CL/P). This study involved a family-based association study of the MTHFR polymorphism. PATIENTS/PARTICIPANTS: We examined 181 patients with CL/P of central European descent and their parents for this variant. RESULTS: The transmission disequilibrium test (TDT) did not confirm an association between the MTHFR 677C-->T polymorphism and nonsyndromic CL/P as previously suggested (p = .36). When comparing the offspring of mothers with periconceptional use of folate to those without, no statistically significant differences were found (p = .708). CONCLUSION: Our data suggest that the MTHFR 677C-->T polymorphism does not make a major contribution to the occurrence of CL/P among central Europeans.

Toll-like receptor 4 ligation enforces tolerogenic properties of oral mucosal Langerhans cells.

BACKGROUND: Despite high bacterial colonization, acute infections are rare in the oral mucosa, implicating tolerogenic predominance. Bacterial antigens like LPSs are recognized by innate immunity receptors such as Toll-like receptor 4 (TLR4), associated with LPS receptor (CD14). OBJECTIVES: Toll-like receptor 4 agonist monosphoryl lipid A has been successfully used as adjuvant in subcutaneous immunotherapy, suggesting reinforcement of allergen-specific tolerance. Recently sublingual immunotherapy (SLIT) has been shown to be an effective alternative to subcutaneous immunotherapy. We observed CD14 expression on human oral Langerhans cells (oLCs), representing a major target of SLIT. However, not much is known about TLR4 expression and its effect on oLCs. METHODS: Cell suspensions were obtained by trypsinization of human oral mucosa and analyzed by flow cytometry, RT-PCR, cytometric bead arrays, ELISA, and mixed lymphocyte reactions. RESULTS: We could show that oLCs express TLR4, and its ligation by monosphoryl lipid A upregulated expression of coinhibitory molecules B7-H1 and B7-H3 while surface expression of costimulatory molecule CD86 was concomitantly decreased. Furthermore, TLR4 ligation on oLCs increased their release of the anti-inflammatory cytokine IL-10 and decreased their stimulatory capacity toward T cells. Moreover, TLR4-ligation on oLCs induced IL-10, TGF-beta1, Forkhead box protein 3, IFN-gamma, and IL-2 production in T cells. CONCLUSION: In view of these data, TLR4-ligation on oLCs might not only play a role in pathogen recognition for efficient immunity but also contribute to the tolerogenic state predominating in the oral cavity.

Prenatal cleft lip and maxillary alveolar defect repair in a 2-step fetal lamb model.

PURPOSE: As there is no satisfying animal model simulating the complex cleft lip and palate anatomy in a standardized defect on one hand, and comprising the possibilities for extensive surgical procedures on the other hand, an improved fetal lamb model for cleft surgery was developed. MATERIALS AND METHODS: Prior to the main study with 16 animals, a pilot study with 4 lambs was conducted. In the pilot study, the unilateral defect was induced at 75 days of gestation. Within 22 days the defect was repaired in 3 lambs; 1 lamb remained unoperated. Disappointing results from the pilot study led to an earlier defect induction (60 to 64 days of gestation) and earlier repair (71 to 84 days) in the main study with 16 lambs. The subsequent delayed repair of the defect was carried out using a Tennison-Randall technique in 10 lambs. In 4 lambs the defect was repaired postnatal, using the same technique. Two lambs had to be excluded from the study. After being euthanized, all animals were investigated macro- and microscopically. RESULTS: According to our criteria, the esthetic results ranged from satisfactory to good. Cutis and mucosa showed a full recovery whereas subcutis and the orbicularis oris muscle showed healing with scar formation. On average the operated lips were 9% shorter and were also thinner than the contralateral control side. CONCLUSIONS: In this study, the results of the closure of a standardized lip and maxillary alveolar defect in several stages of gestation were documented. Early intervention led to better esthetic results, but increased the risk of abortion by 25%. There was no prevention of scarring in subcutaneous and muscle tissue. Because there was no alignment of the orbicularis oris muscle, the goal of a functional perfect result was not achieved.