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Cancer of the oral cavity is known to have a diverse aetiology that includes infectious agents. Human papilloma virus has been found to be associated with several types of human cancer, inclusive of cervical, vulvar, vaginal, penile, anal, and cancer of tonsil. The aim of this manuscript is to investigate the presence of human papilloma virus in tonsillar microbiota of an Afghan population group. A sample of the tonsillar microbiota was collected by oral swab paper stick from 80 healthy donors. The sample was investigated for the presence of high-risk human papillomavirus types 16, 18, 31 and 45 by real time PCR. Eight samples produced some positive endpoint signals for human papillomaviruses. The human papillomavirus 31 was the unique papillomavirus detected; its calculated prevalence rate was 0.10 (C.I. 0.05-0.19). However, the viral load was always very low, in the order of 10-3 viral genomes per cell. The high prevalence of high-risk human papillomavirus in healthy population suggest a need for further investigation on virus spreading and supports the development of vaccination strategies.

Objectives:microRNAs have recently proposed as potential biomarkers in the early detec-tion of Oral Squamous Cell Carcinomas (OSCCs) by noninvasive methods.Aim of the present study was to analyze the expression of a panel of miRNAs in epithelialcells collected by oral brushing from OSCCs and their respective normal mucosa in distantareas and from regenerative areas after OSCC surgical resectionMethods:Oral brushing specimens were collected from: 13 OSCC (group 1a) and theirrespective normal mucosa in distant areas (group 1b), 12 samples from regenerative areasafter OSCC surgical resection (group 2a) and their respective normal mucosa in distantareas (group 2b), 17 lichenoid lesions (group 3) and 24 normal mucosa from healthy con-trols (group 4). In all different groups the expression levels of miRNAs were evaluated byreal time PCR. Seven target were evaluated (i.e. mir-21, miR-375, miR-345, miR-181b,miR146a, miR-649, miR-518b). The miRNA expression levels across groups wereassessed by ANOVA statistics or student t test for paired samplesResults:miR345, miR146, miR649 e miR518 showed a significant different expression inOSCC group with respect to healthy donors, but a similar and not significant expressionwith respect to their respective normal mucosa in distant areas (group 1b). MoreovermiR146 showed a significantly different expression in regenerative areas after OSCC sur-gical resection with respect to healthy donors, but not significant difference with respect totheir respective normal mucosa in distant areas. No significant differences were foundbetween lichenoid lesions and healthy donors in any miRNAs analyzed.Conclusions:miRNA analysis in epithelial cells collected by oral brushing has demon-strated the presence an altered profile expression not only in OSCC but also in distantareas from OSCC and in regenerative areas after OSCC surgical resectionRelevance:Analysis of miRNA expression from oral brushing could be proposed as anon invasive method to study oral mucosa in OSCC patients.

Background: Periodontitis is a disease that affects and destroys the tissues that support teeth. Tissue damage results from a prolonged inflammatory response to an ecological shift in the composition of subgingival biofilms. Three bacterial species that constitute the red complex group, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, are considered the main pathogens involved in periodontitis. Materials and Methods: In the present study, a real-time polymerase chain reaction bases assay was designed to detect and quantify red complex species, then used to investigate 307 periodontal pocket samples from 127 periodontitis patients and 180 controls. Results: Significant higher prevalence of red complex species and increased amount of P. gingivalis and T. denticola were detected in periodontal pocket of periodontitis patients. Conclusions: Results demonstrated that the test is a valuable tool to improve diagnosis of periodontal disease.

Peri-implantitis is a destructive inflammatory process that affects the tissues around osseointegrated implants, resulting in the formation of a pen-implant pocket and loss of the supporting bony (1). Peri-implantitis are observed in 12-43% of the implants (2). The most evident clinical signs are hyperplasia of the soft tissues, suppuration, gradual bone loss and progressive mobility of the implant. One of the causes of the peri-implantitis is the bacterial colonization of implant surfaces. Lipopolysaccharides from the cell walls of periodontal pathogens induce the release by monocytes and macrophages of pro-inflammatory mediators like cytokines and tumor necrosis factor alpha. These mediators stimulate fibroblasts to produce prostaglandins and metalloproteinases, which determine the decomposition of alveolar bone and extracellular matrix destruction. In these cases, the formation of a granulation tissue can replace the bone gradually resorbed. The progression of the disease frequently leads to osteomyelitis followed by the loss of the implant after few weeks. However, the disease progression is influenced by the host response to bacterial toxins. This response depends by different factors like environmental (poor oral hygiene, smoking, stress), systemic (diabetes, osteoporosis) and genetic (polymorphisms in genes encoding molecules of the host immune defense). Microbiota associated to healthy pen-implant tissue is composed by gram-positive bacteria like Streptococcus mitis, Streptococcus sanguis and Streptococcus oralis. These microrganisms create a series of prior conditions for the adhesion of periodontal pathogens, being able to induce the development of peri-implantitis (3). The microbiota associated with failure of the implants is very similar to that found in periodontal disease and is characterized by high counts and proportions of gram-negative anaerobic bacteria. The principal microrganisms involved in penimplantitis are members of the red complex species (Poiphyromonas gingivalis, Treponema denticola and Tannerella forsythia) and orange complex species (Fusobacterium sp. and Prevotella intermedia), as defined by Socransky et al. (4). Hultin et al (5) recovered high count of A ctinobacillus actinomycetemcomitans and Aggregatibacter actinomycetemcomitans around implants with peri-implantitis. Staphylococcus aureus, enteric rods and Candida albicans, are also associated with pen-implant infections (6-8). Alcoforado et al. (9) microflora consisting Fuso bacterium species, Candida albicans. Leonhardt et al. (10) found that peri-implantitis sites are invaded by Poiphyromonas gingivalis, Porphyronionas intermedia, and Actinobacillus aclinomycetemcornitans. These pathogens were observed in ITT® and Brânemark fixtures at 3 to 6 months following implant placement (11). identified a diseased implant of Campylobacter rectus, Prevotella intermedia and fixtures at 3 to 6 months following implant placement (11)

Recent evidence indicates that microRNAs affect cell growth, differentiation, apoptosis, and play a role in tumorigenesis. Large scale screening suggested that mir-146a was related to oral squamous cell carcinoma development and progression. The common polymorphism rs2910164, able to modulate mir-146a expression appeared related to the onset of different cancers, including carcinoma. A sample set of 360 oral squamous cell carcinoma was genotyped to test association between rs2910164 and cancer occurrence as well as cancer progression. No significant levels of association with any allele or genotype was found, however a slight increase of the variant allele was observed in stage II tumors. Further studies could help to understand if rs2910164 may considered a risk factor for oral carcinoma.

Legionella spp. are ubiquitous in aquatic habitats and water distribution systems, including dental unit waterlines. Surveys have shown that the percentage of samples taken at different dental sites that were positive for Legionella spp. was highly variable and ranged from 0% to 100%. Cultivation is the principal approach to evaluating bacterial contamination employed in the past, but applying this approach to testing for Legionella spp. may result in false-negative data or underestimated bacterial counts. PCR and direct fluorescent counts can detect viable non-cultivable bacteria, which are not counted by plating procedures. Legionella spp., commonly form such viable non-culturable cells and it is likely that they contribute to the difference between plate count results and those of PCR and fluorescent-antibody detection. However, studies have shown that Legionella is present in the municipal water source in spite of the current filtration and chlorination procedures. Once Legionella reaches the building water system, it settles down into a biofilm layer of stagnant water. By means of this layer, Legionella can protect itself from antimicrobial agents and then multiply. Dental unit waterlines may be contaminated with opportunistic bacteria. The water quality in the dental units should be controlled to eliminate opportunistic pathogens and to provide water for dental treatment that meets public health standards for potable water. Legionella spp. are ubiquitous in aquatic habitats and water distribution systems, including dental unit waterlines. Surveys have shown that the percentage of samples taken at different dental sites that were positive for Legionella spp. was highly variable and ranged from 0% to 100%. Cultivation is the principal approach to evaluating bacterial contamination employed in the past, but applying this approach to testing for Legionella spp. may result in false-negative data or underestimated bacterial counts. PCR and direct fluorescent counts can detect viable non-cultivable bacteria, which are not counted by plating procedures. Legionella spp., commonly form such viable non-culturable cells and it is likely that they contribute to the difference between plate count results and those of PCR and fluorescent-antibody detection. However, studies have shown that Legionella is present in the municipal water source in spite of the current filtration and chlorination procedures. Once Legionella reaches the building water system, it settles down into a biofilm layer of stagnant water. By means of this layer, Legionella can protect itself from antimicrobial agents and then multiply. Dental unit waterlines may be contaminated with opportunistic bacteria. The water quality in the dental units should be controlled to eliminate opportunistic pathogens and to provide water for dental treatment that meets public health standards for potable water.

The aim of the present study is to evaluate the peri-implant soft tissues and the amount of inflammatory cells around two different implant-abutment connections (self-locking conical connection with platform switching and screwed connection with standard abutment and internal anti-rotational system). Histological analysis was made of 14 implants, 7 with self-locking Morse tapered connection (experimental group A) and 7 with screw-retained anti-rotational connection (control group B). Sixty days after non-functional immediate loading, peri-implant tissue biopsies were performed. In the samples taken from the experimental group the peri-implant connective tissue consisted of a greater density of collagen and fibroblasts compared to the connective tissue of the control group. The experimental group specimens showed less inflammatory infiltrate close to the self-locking tapered connection compared to the tissues around the screw-retained connection. The SEM observations showed less microgap in the self-locking conical connection than in the screw connections with standard abutment and internal anti-rotational system. The presence of connective tissue with few inflammatory cells and the absence of inflammatory infiltrate, in self-locking conical connection implants is due to the minimal size of the implant-abutment microgap that does not allow the passage of fluids and bacteria from the oral cavity to the implant thus preventing tissue inflammation. Copyright © by BIOLIFE, s.a.s.

Demineralized freeze-dried bone allograft (DFDBA) is widely used in periodontal regeneration procedures as a scaffold for new bone formation in periodontal defects. How this biomaterial alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to DFDBA. By using DNA microarrays containing 20,000 genes, the authors identified in an osteoblastlike cell line (MG-63) cultured with DFDBA (Allogro, Dentsply/Friadent-Ceramed) several genes whose expression was significantly up-regulated or down-regulated. The differently expressed genes cover a broad range of functional activities: (1) cell cycle regulation, (2) immunity, (3) vesicular transport, (4) production of cytoskeletal elements, and (5) bone remodeling. The data reported are, to the authors' knowledge, the first genetic portrait of DFDBA effects. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Periodontal tissues surround the teeth and provide their attachment. Periodontal diseases include a mild and reversible form named gingivitis, and periodontitis that is the main cause of tooth loss in adults. Gingivitis, that affects gums and coronal junctional epithelium, as well as periodontitis, that is characterized by loss of connective tissue attachment, are caused by a persistent inflammatory response promoted by alteration of periodontal biofilm. The aim of the study was to test whether the prevalence or relative amount of each species was associated with a particular clinical condition. Periodontal evaluation of 539 unrelated patients was performed by the Periodontal Screening and Recording (PSR) system. Subgingival samples were obtained from the site with the worst PSR score. A selection of eleven bacterial species was evaluated by quantitative real time PCR. Some bacterial species were found to be associated with all phases of periodontal disease, such as Tannerella forsythia, Treponema denticola, and Treponema lecithinolyticum, while other species were more specifically associated with periodontitis, such as Porphyromonas endodontalis and Porphyromonas gingivalis, or with gingivitis, such as Capnocytophaga ochracea and Campylobacter rectus. Quantitative and qualitative analyses helps to better understand the microbial changes associated with different stages of periodontal disease.

Bio-stimulation is a technique in aesthetic medicine in which different drugs such as nucleotides, antioxidants and glucosaminoglycans precursors are injected in the dermis to improving the anabolic function of dermal fibroblasts, i.e., protein synthesis, replication and production of extracellular matrix components. It can be achieved with multiple intra-dermal injections, using two protocols: 1) Polydeoxyribonucleotide (PDRN) plus glucosamine sulphate (Gluc); 2) N-acetylcysteine (NAC) and amino acids (Aa) (named Bio- NAC procedure). Since the role of drugs used in biostimulation on human dermal fibroblasts is not completely understood, the aim of this study is to evaluate the effect of these substances in primary cell cultures by using RT-PCR and a panel of specific genes (ELN, DSP, FN1, FBN1, ITGA1, ITGA2, ITGA5, ITGB1, COL1A1,COL3A1) to detect their effect on cell metabolism and extracellular matrix components. Both the treatments were responsible for Elastine and Desmoplakin genes activation. Only NAC plus Aa treatment enhance the expression of other genes related to tissue growth and elasticity like FBN1, ITGA1 and ITGB1. All the other genes investigated (FN1, ITGA5, ITGA2, COL1A1, COL3A1) were down-regulated by both treatments. Since the precise role of these proteins in tissue integrity and aging is not known, this study confirms the usefulness of biostimulation therapies in enhancing some of the genes responsible of cellular wellbeing. This study could be useful to consider the possibility of injective biostimulation in oral cavity, clinical applications in oral healing and in gingival atrophy as well.

Epithelial to Mesenchymal Transition (EMT) is an important process involved in cancer, embryogenesis and organ development. Its role in nonsyndromic cleft lip with or without cleft palate (NSCL/P) has been extensively investigated and successfully linked to the disease. In this study, we focused on a gene, CDH1, encoding for E-cadherin, a key protein in EMT. We carried out an association study on an Italian sample group, genotyping four single nucleotide variations within the CDH1 gene, in order to verify the potential role of this gene in NSCL/P etiology. Neither the haplotype nor the family-based association test revealed any association between the genotyped SNPs and the pathology. Our results demonstrate that, in our Italian sample study, the analyzed single nucleotide polymorphisms are not associated to NSCL/P.

Polyamines are involved in normal and pathological cell proliferation and differentiation. Like acid radicals, they play an important role in remodelling the extracellular matrix and are considered "uremic toxins". Proteins and glycosaminoglycans are essential components of the extracellular matrix, and contribute to normal mature organ functions. The aim of this study was to analyse the effects of free polyamines, dialysate fluid components and dialysis fluid on protein and extracellular glycosaminoglycan synthesis in VERO cell cultures.The dialysate fluid components were separated with a Sephadex G15 column and the cultures were analysed after incorporation of 3H-leucine and 3H-glucosamine. Cultures were run at pH 7.0 and pH 7.4. The glycosaminoglycan classes were separated with a DEAE column, and polyamines were determined by high-performance liquid chromatography. Proteins and single glycosaminoglycan classes were quantified by a scintillator. DNA gel electrophoresis was done to detect chromatin fragmentation.Dialysate contained putrescine, spermidine and spermine, chromatography showing four peaks; only peaks I and II indicated polyamines at respectively Da 5000 and 1500. Polyamines are therefore linked to different carriers. There was an increase of protein and glycosaminoglycan synthesis with dialysis fluid and polyamines, but inhibition with peak II or dialysate. DNA gel electrophoresis showed no chromatin fragmentation. Findings at pH 7.0 and 7.4 were similar.It would appear that in uremic patients polyamines are conjugated to protein carriers of different molecular weights with different biological actions. As polyamines and dialysis fluid affect changes in extracellular matrix, they could be related to physiological organ functions. However, these in vitro data must be considered with the appropriate limitations when we try to extrapolate them to the in vivo situation.

Background: Craniofacial morphogenesis is the result of an intricate multistep network of tightly controlled spatial and temporal signalling that involves several molecules and transcription factors organized into highly coordinated pathways. Any alteration in even one step of this delicate process can lead to congenital malformations such as cleft palate. One of the first steps in embryonal orofacial development is the migration of cells from the neural crests to the branchial arches. Next, the cells have to proliferate, differentiate, move and connect to each other in order to correctly form the palate. Cell contraction, promoted by the interaction of non-muscle myosin II and actin A, is a crucial step in morphogenesis and is regulated by ROCK1 protein. Methods: A family-based association study was carried out in order to verify whether or not genetic variants of ROCK1 were associated with non-syndromic cleft palate (nsCP). Two cohorts from Italy and Iran, a total of 189 nsCP cases and their parents were enrolled. Results: The rs35996865-G allele was under-transmitted in cases of nsCP [P=.006, odds ratio (OR) = 0.63 (95% CI 0.45-0.88)]. Conclusion: This investigation reveals for the first time data supporting a role for ROCK1 in nsCP aetiology.

Gabapentin is one of the most used drugs to treat postoperative pain with antihyperalgesic properties and has a unique mechanism of action, which differentiates it from other commonly used drugs. Various studies have shown that the perioperative use of gabapentin reduces postoperative pain. In our study, fragments of gingival tissue of healthy volunteers were collected during operation. Gene expression of 29 genes was investigated in gingival fibroblasts cell culture treated with gabapentin, compared with untreated cells. Of the different chemokines and interleukins studied, only 10 were statistically significant (CCL1, CCR1, CCR4, CCR5, CCR6, ILI1A, ILI1B, IL5, IL6R, TNFSF10). The overexpression of these cytokines, obtained in many studies, leads us to think that gabapentin can interact and cause post-inflammatory gingival hyperplasia, but, probably, in our study the gabapentin has not the same effect, because we used gingival fibroblasts of healthy people.

Diagnosis of focal disease, the theory that the human oral microbial (HOM) could affect the onset and development of systemic diseases, was very popular in the past, but the lack of scientific evidence has led to the abandonment of this idea. Interestingly, increasing evidence over the past 3 or so decades suggests that HOM can indeed serve as a reservoir for systemic dissemination of pathogenic bacteria and their toxins in distant body sites, favouring the developments of malignant tumours. Malignant tumours are complex communities of oncogenically transformed cells with aberrant genomes, associated nonneoplastic cells including immune and stromal cells, and sometimes HOM, including bacteria and viruses. Recent data suggest that HOM and periodontal disease play an active role in the pathogenesis of colorectal cancer, in fact HOM has been found within the colorectal cancer microenvironment, and the composition of the HOM was different from that of adjacent non- neoplastic tissue. An association of fusobacterium nucleatum with the colonic mucosa of colorectal cancer has been proven. Several questions thus arise. Is periodontal disease a risk factor for colorectal carcinoma? Given the connectivity of the digestive tract, could fusubacterium nucleatum or other HOM be involved in additional gastrointestinal disorders? Furthermore, based on the “mobility” of Fusubacterium nucleatum and the omnipresence of cadherins, could this organism be involved in cancers beyond the gastrointestinal tract? Answers to these questions will shed new lights on the role of the HOM in onset of diseases.

Harvesting bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. However, as stem cells are characterized by self-renewing and commitment in several cellular subtypes (ie, pluripotential differentiation), some concerns may arise as regards their potential uncontrolled proliferation. To screen the behavior of osteoblasts derived from human pulpar stem cells (ODHPSCs), we used microarray techniques to identify genes that are differently regulated in ODHPSC in comparison to normal osteoblasts (NOs). Osteoblasts derived from human pulpar stem cells were obtained from human dental pulp, and cells were selected using a cytometer. The cell profile was c-kit+/CD34+/STRO-1+/CD45−. These cells were capable of differentiation of osteoblasts in vitro. By using DNA microarrays containing 19,200 genes, we identified in ODHPSC some genes whose expression was significantly up- and downregulated compared to NO. The differentially expressed genes have different functional activities: (a) cell differentiation, (b) developmental maturation, (c) cell adhesion, and (d) production of cytoskeleton elements. Thus, some molecular differences exist between NO and ODHPSC, although the previously considered histologic parameters show a normal phenotype.

Central giant cell granuloma (CGCG) of the jaws is a central osteolytic lesion characterized histologically by multinucleated giant cells in a background of ovoid to spindle-shaped mesenchymal cells. Whether CGCG is a reactive lesion or a truly benign neoplasm remains undetermined, and the mechanism determining the onset of the disease remains unknown. To have more information regarding the genetic events involved in CGCG, the authors decided to perform an expression profile. Samples were derived from two surgically resected CGCG of the mandible. RNA extracted from a pool of three normal bone tissues was used as control. By using DNA microarrays containing 19,200 genes, the authors identified several genes whose expression was significantly up- or down-regulated. The differentially expressed genes cover a broad range of functional activities: cell cycle regulation; signal transduction; and vesicular transport. It was also possible to detect some genes whose function is unknown. The authors believe the data reported to be the first genetic portrait of CGCG of the jaws. Several markers have been identified that can potentially help in identifying some biological behavior (ie, quiescent versus aggressive lesions), as well as genes whose products could be potentially disease-specific targets for therapy. However, the authors think that more cases are needed, especially those comparing quiescent and aggressive lesions, before the exact profile of CGCG is known.

Granular cell tumor (GCT), or granular cell myoblastoma, is a relatively uncommon lesion of the soft tissues. It can occur in any organ, and the tongue is more often affected. GCT has unknown etiology, uncertain histogenesis, and a not always benign nature. Benign myoblastomas are the great majority, but rare malignant lesions have been reported. To have more information regarding the genetic events involved in GCT, the authors decided to perform an expression profile. A sample was derived from a surgically resected GCT of the tongue. RNA extracted from normal tongue (mucosa plus muscle) was used as control. By using DNA microarrays containing 19,200 genes, the authors identified several genes for which expression was significantly up- or down-regulated. The differentially expressed genes cover a broad range of functional activities: (1) signal transduction, (2) cell cycle regulation, and (3) cytoskeleton organization. It was also possible to detect some genes whose function is unknown. The data reported are, to the authors' knowledge, the first genetic portrait of GCT. Mutations in some of the described genes are related to neural alterations and mental diseases, and this fact supports the idea of a neural origin of myoblastoma. Several markers have been identified that will help in identifying the biological behavior (when malignant lesions will be described), as well as the gene whose products could be potentially disease-specific targets for therapy.

Nonsyndromic cleft lip with or without cleft palate (CL/P) affects approximately 1 in 1,000 births. Genetic studies have provided evidence for the role of several genes and candidate loci in clefting; however, conflicting results have frequently been obtained and much have to be done to unravel the complex genetics of CL/P. In the present investigation we have focused on the candidate region in 6p23, a region that have been found linked to CL/P in several investigations, in the attempt to find out the susceptibility gene provisionally named OFC1. Gene expression experiments in mice embryo of positional candidate genes revealed that JARID2 was highly and specifically expressed in epithelial cells in merging palatal shelves. A family-based linkage disequilibrium study confirmed the pivotal role of JARID2 in orofacial development and strongly supports a role for this gene in CL/P etiology (multiallelic haplotype test P=6×10−5). Understanding the molecular role of JARID2 within facial development may offer additional information to further unravel the complex genetics of CL/P. Hum Mutat 31:1–7, 2010. © 2010 Wiley-Liss, Inc.

Clefts of the orofacial region are among the most common facial defects and are caused by abnormal facial development during gestation. Cleft lip with or without cleft palate (CL/P) is a birth defect with a complex etiology resulting from a mixture of genetic and environmental factors. In the present study we considered myosin 14 (MYH14) as a candidate gene for CL/P. This gene codes for the heavy chain of non-muscle myosin IIC (NMMHC-IIC), maps in the OFC3 region, and shares significant homology with myosin 9, a gene that our group has recently seen to be involved in CL/P. A linkage disequilibrium investigation was conducted with six single nucleotide polymorphisms in MYH14 and a sample of 239 CL/P nonsyndromic patients and their parents. Our family-based investigation provided no evidence of association between MYH14 and CL/P alleles. These data do not support the involvement of MYH14 in CL/P among the Italian population.

Genetic studies have demonstrated that nonsyndromic cleft is composed of two separate entities: the cleft palate only and cleft of the lip, alveolus with or without cleft palate; both have a heterogeneous genetic background and environmental factors contribute to the onset of these malformations. The role of transforming growth factor alpha (TGF-A) was considered possible, but conflicting results have been reported. To detect if TGF-A is involved in the onset of cleft diseases, a series of patients with nonsyndromic clefts and control subjects were analyzed with regard to protein expression. Forty-three patients with nonsyndromic clefts and 21 unaffected subjects were enrolled in this study. Paraffin-embedded specimens were matched with TGF-A antibody and then scanned with a computerized image analyzer. TGF-A was scored as absent, moderately (from 10% to 30%), and highly expressed in epithelium, gland, and muscle. Data were statistically analyzed with a Kruskal-Wallis test. Comparison between control subjects and patients with clefts showed that only gland and epithelium reached a significant P value. A subsequent comparison between cleft of the lip, alveolus with or without cleft palate and cleft palate only groups demonstrated a statistically significant difference only for gland. TGF-A was decreasingly expressed in unaffected, cleft of the lip, alveolus with or without cleft palate, and patient with cleft palate only and thus further strength has been given to its role in the onset of the disease. © 2007 Muntaz B. Habal, MD.

Non-syndromic cleft lip with or without palate (CL/P) is one of the most common malformations among live births, but most of the genetic components and environmental factors involved remain to be identified. Among the different causes, MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA, was considered a potential candidate, because it was found to be abundantly and specifically expressed in epithelial cells of palatal shelves before fusion. After fusion, its expression level was shown to decrease and to become limited to epithelial triangles before disappearing, as fusion is completed. To determine whether MYH9 plays a role in CL/P aetiology, a family-based association analysis was performed in 218 case/parent triads using single-nucleotide polymorphism (SNP) markers. Pairwise and multilocus haplotype analyses identified linkage disequilibrium between polymorphism alleles at the MYH9 locus and the disease. The strongest deviation from a null hypothesis of random sharing was obtained with two adjacent SNPs, rs3752462 and rs2009930 (global p value = 0.001), indicating that MYH9 might be a predisposing factor for CL/P, although its pathogenetic role needs to be investigated more accurately.

Background: Genetic studies have demonstrated that non-syndromic cleft is composed of two separate entities - cleft palate only (CPO) and cleft of lip, alveolus with or without cleft palate (CL ± P) -, both have a heterogeneous genetic background and environmental factors contribute to the onset of these malformations. Previous studies have shown that TGFβ3 could be involved in these diseases, but no conclusive results have been reached. Purpose: In order to detect if TGFβ3 has a role in cleft diseases, a series of non-syndromic cleft patients and controls are analyzed for TGFβ3 protein expression. Material and methods: Forty-three non-syndromic cleft patients and 21 unaffected subjects were involved in this study. Paraffin-embedded specimens were matched with the TGFβ3 antibody and then scanned with a computerized image analyzer. TGFβ3 was found to be absent (less than 10%), moderate (from 10% to 30%) and highly expressed (higher than 30%) in epithelium (EP), minor palatal salivary gland (GL) and fibres of elevator palati muscle (MU). Data was statistically analyzed with a Kruskal-Wallis test. Results: Only GL and EP have a statistically significant lower expression in non-syndromic cleft compared to unaffected subjects. A subsequent comparison between CL ± P and CPO groups demonstrates a statistically significant difference only for GL, with a lower expression in GL of CPO patients. Conclusions: TGFβ3 is decreasingly expressed in GL of unaffected CL ± P and CPO patients and thus further strength is given to a pathogenetic role of TGFβ3 in the onset of clefts. © 2006 Elsevier Ireland Ltd. All rights reserved.

Folate receptor family members (FOLRs) mediate the delivery of 5-methyltetrahydrofolate to the interior of, out of within, or between cells in a process known as potocytosis. Three FOLRs and a pseudogene map to 11q13.4. The aim of this study was to verify whether FOLRs could be responsible for the onset of nonsyndromic cleft lip with or without cleft palate (CL/P). Linkage and linkage disequilibrium between genetic markers and disorder were analyzed. Patients and their mothers from 71 familial CL/P pedigrees and 75 sporadic cases from Italian population were investigated by PCR-SSCP analysis. Data from mutation scanning allowed us to find only a silent mutation in FOLR1 present in a mother and her child. Our findings do not support FOLR1 and FOLR2 genes in the onset of CL/P.

Calcium sulfate (CaS) is an highly biocompatible material that has the characteristic of being one of the simplest as well as one of the synthetic bone-like graft with the longest clinical history, spanning more than 100 years. Solidified or crystallized CaS is very osteogenic in vivo. As the surface CaS dissolves in body fluid, the calcium ions form calcium phosphate that reprecipitates on the surface forming an osteoblast "friendly" environment. How this "friendly" environment alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identified genes that are differently regulated in osteoblasts exposed to CaS. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) cultured with CaS (Surgiplaster, Classimplant, Roma, Italy) several genes that expression was significantly upregulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) lysosomal enzymes production, (c) cell cycle regulation, (d) and signaling transduction. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of CaS effects. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Oral cavity and oropharynx cancer associated with human papil loma virus infection, particularly in young people who are continuously exposed to this virus, is a serious public health problem worldwide, especially for high-risk strains that are most associated with premalignant lesions and tumors. These neoplasms remain asymptomatic for a long time and, when they occur, they are already at an advanced stage. If diagnosed and treated early, oral cancer induced by human papilloma virus allows for high survival, as it often has a more favorable prognosis than oral cancers not directly related to viral infection. In this study, the presence of different high-risk and low-risk HPV strains was investigated to assess the epidemiological status in a population of healthy individuals. Two types of samples, one from the tonsils and one from the base of the tongue, were collected from 2015 healthy individuals of different sex and age. A total of 1885 DNA samples belonging to 1285 patients were tested for the presence of 11 high-risk HPV types plus 4 low-risk HPV types using real-time PCR. Of the patients’ DNA samples screened for 15 HPV types, only four samples were positive, all of which were taken from male smokers. These results indicate that newly acquired oral oncogenic HPV infections in the healthy population are rare and, in many cases, controversial. Therefore, more studies are needed to ensure fewer variations in outcomes and a greater clarification of HPV infection and its prevalence in the oropharynx of the healthy population, and to guide efforts to prevent the development of this infection which, if undiagnosed, can lead to the onset of malignant tumors in the oral cavity.

In recent decades, many efforts have been made to elucidate the genetic causes of non-syndromic cleft palate (nsCPO), a complex congenital disease caused by the interaction of several genetic and environmental factors. Since genome-wide association studies have evidenced a minor contribution of common polymorphisms in nsCPO inheritance, we used whole exome sequencing data to explore the role of ultra-rare variants in this study. In a cohort of 35 nsCPO cases and 38 controls, we performed a gene set enrichment analysis (GSEA) and a hypergeometric test for assessing significant overlap between genes implicated in nsCPO pathobiology and genes enriched in ultra-rare variants in our cohort. GSEA highlighted an enrichment of ultra-rare variants in genes principally belonging to cytoskeletal protein binding pathway (Probability Density Function corrected p-value = 1.57 × 10−4); protein-containing complex binding pathway (p-value = 1.06 × 10−2); cell adhesion molecule binding pathway (p-value = 1.24 × 10−2); ECM-receptor interaction pathway (p-value = 1.69 × 10−2); and in the Integrin signaling pathway (p-value = 1.28 × 10−2). Two genes implicated in nsCPO pathobiology, namely COL2A1 and GLI3, ranked among the genes (n = 34) with nominal enrichment in the ultra-rare variant collapsing analysis (Fisher’s exact test p-value < 0.05). These genes were also part of an independent list of genes highly relevant to nsCPO biology (n = 25). Significant overlap between the two sets of genes (hypergeometric test p-value = 5.86 × 10−3) indicated that enriched genes are likely to be implicated in physiological palate development and/or the pathological processes of oral clefting. In conclusion, ultra-rare variants collectively impinge on biological pathways crucial to nsCPO pathobiology and point to candidate genes that may contribute to the individual risk of disease. Sequencing can be an effective approach to identify candidate genes and pathways for nsCPO.

Objective: To evaluate the subgingival microbiological changes during the first six months of therapy with clear aligners (CAs) and fixed appliances (FAs). The null hypothesis was that there would be no microbiological differences between the two. Setting/Sample: Two groups of patients to be treated, respectively, with CAs (14 patients; 9 females and 5 males; mean age 21years±0.25) and FAs (13 patients; 8 females and 5 males; mean 14years±0.75) were consecutively recruited. Materials and Methods: Subgingival microbiological samples were obtained at the right upper central incisor and right first molar at four different time points: before appliance fitting (T0), and at 1month (T1), 3months (T3) and 6months (T6) thereafter. Total bacterial load (TBL) and counts of the bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Treponema denticola and Tannerella forsythia were determined using real-time PCR. Results: Total bacterial load did not vary in the CA group, while a significant increase was detected after 3 and 6months of treatment in the FA group. Unlike red complex species, Crectus and Fnucleatum were often detected: levels remained stable in the CA group but increased progressively in the FA group. Conclusion: The type of orthodontic appliance influences the subgingival microbiota. TBL increased in the FA group but not in the CA group, although the levels of the individual periodontal pathogenic bacteria species did not significantly increase during the observation period.

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a frequent orofacial malformation. The comparison of concordance rate observed in monozygotic and dizygotic twins supports high level of heritability and a strong genetic component. However, phenotype concordance for orofacial cleft in monozygotic twins is about 50%. The aim of the present investigation was to detect postzygotic events that may account for discordance in monozygotic twins. High-density SNP microarrays hybridization was used to genotype two pairs of monozygotic twins discordant for nsCL/P. Discordant SNP genotypes and copy number variants were analyzed to identify genetic differences responsible of phenotype discrepancy. A number of differences were observed, none involving known nsCL/P candidate genes or genomic regions. Considering the limitation of the study, related to the small sample size and to the large-scale investigation method, the results suggest that the detection of discordant events in other monozygotic twin pairs would be remarkable and warrant further investigations.

Orofacial clefts are common congenital defects whose prevalence differs between geographical regions and ethnic groups. The inheritance is complex, involving the contribution of both genetic and environmental factors. The involvement of genes belonging to the folate pathway is still matter of debate, with strong evidences of association and conflicting results. After demonstrating the contribution, for a sample from the Italian population, of common mutations mapping on three genes of the folate pathway, our group tried to unravel their contribution in independent sample studies with different ethnicity. In the present investigation a set of 34 triads with oral cleft from Nassiriya, Iraq, has been genotyped for rs1801133 of MTHFR, rs1801198 of TCN2, and rs4920037 of CBS polymorphisms. Association analysis evidenced a decreased risk of cleft for children carrying the 667G allele at TCN2 gene ( P = 0.02). This evidence further supported the relationship between polymorphisms of folate related genes and oral clefts, and outlined the relevance of studying populations having different ethnicity.

Peri-implantitis is an inflammatory disease of tissues surrounding osseointegrated dental implants. Inflammation affecting soft and hard peri-implant tissues can cause alveolar bone resorption and subsequent implant loss. Clinical surveillance and early diagnosis are of paramount importance to reduce clinical failures and improve implant survival. Current diagnosis of implants is based on clinical and radiological signs. Molecular tests are an emerging diagnostic methodology, which potentially can help to detect and prevent early peri-implantitis and monitor the efficacy of therapy as well. A plethora of potential biomarkers are potentially available to support the clinical diagnosis of peri-implantitis. However, conflicting diagnostic conclusions have been reached, probably related to weak statistical results due to limited sample size or disease heterogeneity. The present paper reviews candidate diagnostic biomarkers for peri-implantitis, including infective agents, genetic susceptibility factors, and key proteins related to inflammation and tissue remodeling.

Periconceptional folic acid supplementation can reduce the risk of inborn malformations, including orofacial clefts. Polymorphisms of MTHFR, TCN2, and CBS folate-related genes seem to modulate the risk of cleft lip with or without cleft palate (CL/P) in some populations. CL/P and cleft palate only (CPO) are different malformations that share several features and possibly etiological causes. In the present investigation, we conducted a family-based, candidate gene association study of non-syndromic CPO. Three single nucleotide polymorphisms, namely, rs1801133 of MTHFR, rs1801198 of TCN2, and rs4920037 of CBS, were investigated in a sample that included 129 Italian and 65 Asian families. No evidence of association between the three genotyped polymorphisms and CPO was found in the Italian and Asian cases, indeed the transmission disequilibrium test did not detect any asymmetry of transmission of alleles. This investigation, although with some limitation, further supports that CL/P and CPO diverge in their genetic background.

Gene expression and cell behavior are regulated by several factors, including small non-coding RNAs. MicroRNAs affecting cell growth, differentiation, and apoptosis are thought to play an important role in tumorigenesis. The levels of miR-146 appear to be associated with cancer development and progression, including that of oral squamous cell carcinoma. The aim of this investigation was to ascertain whether the single nucleotide polymorphism, rs2910164, mapping in the MIR146A gene, has a role in oral squamous cell carcinoma progression. A genetic association study was performed with a sample set of 346 oral squamous cell carcinomas collected in Italy. Our data indicate that the rs2910164 polymorphism is not associated with tumor development. However, a slight increase in the frequency of the variant allele was observed in Stage II tumors. Further investigations are needed to verify a possible role of the variant allele or rs2910164 in oral squamous cell carcinoma progression.

Studies aimed at evidencing genetic causes for neural tube defect (NTD) occurrence have often provided the inspiration for orofacial cleft aetiology investigations. The correlation between the two congenital malformations is provided by the similar incidence timing and the involvement of structures localized in the midline of the embryo. This connection is corroborated by the existence of a number of genes involved in both malformations. In this article, we considered the dihydrofolate reductase ( DHFR ) gene, previously seen implicated in NTDs, as a candidate for cleft lip with or without cleft palate (CL/P) risk. Four SNPs mapping on the DHFR gene were genotyped for 400 Italian CL/P triads, using TaqMan ® approach. The rs1677693 provided evidence of association, even if at borderline level ( P value 0.049). In particular, the variant allele seems to have a protective effect OR = 0.80 (95% C.I. 0.64–0.99). Moreover, the combination of rs1677693(A)-rs1650723(G) alleles showed a significant association OR 0.64 (95% C.I. 0.47–0.86) ( P value = 0.006). This represents the first attempt to demonstrate a role for DHFR in CL/P aetiology, howbeit the study of such gene deserves a deepening.

Unraveling of factors involved in multifactorial diseases is a great challenge. Different approaches can be contemplate and applied to a variety of congenital malformations. In the present investigation TFAP2A has been considered a good candidate gene for nonsyndromic cleft lip with or without cleft palate (NSCLP) aetiology, basing on a sum of considerations. TFAP2A has been seen involved in orofacial development in mice; it is located in the NSCLP candidate region 6p24; it codes for a transcription factor which regulates expression of IRF6, a gene implied in NSCLP; finally, it is embroiled in the branchiooculofacial syndrome, that includes clefting as feature. A family based association analysis was performed with a sample study of 405 NSCLP triads. Evidence of association was obtained with both single marker and haplotype analyses, thus providing a support for TFAP2A in NSCLP aetiology.

Several investigations have reported that the human papillomavirus may play a role in head and neck squamous-cell carcinoma development and may have a prognostic impact. However, inconsistent results regarding human papillomavirus prevalence have been obtained so far. Variables which may account for these discrepancies may be related to site of the samples' origin, detection methods and sample size. The aim of this study is to evaluate the presence of high-risk type human papillomavirus in a large, well defined sample of squamous-cell carcinoma limited to the oral cavity in its strictest definition-ie with no pharynx or larynx cancers included-by means of quantitative real-time polymerase chain reaction, a method which ensures high sensitivity and offers the opportunity to exclude false-positive results. Data were obtained from 314 squamous-cell carcinoma limited to oral cavity proper, indicated that the prevalence of high-risk human papillomavirus was as low as 2% (CI 0.6-3). Matched pairs case-control analysis indicated that the prevalence among controls did not significantly differ with respect to cases and thus did not support a major role for the human papillomavirus in the etiology of squamous-cell carcinoma of the oral cavity.