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Extracellular vesicles (EVs) are mediators of the immune system response. Encapsulated in EVs, microRNAs can be transferred between cancer and immune cells. To define the potential effects of EVs originated from squamous cell carcinoma cells on immune system response, we performed microRNA profiling of EVs released from two distinct cell lines and treated dendritic cells derived from circulating monocytes (mono-DCs) with these EVs. We confirmed the internalization of EVs by mono-DCs and the down-regulation of microRNA mRNA targets in treated mono-DCs. Differences in surface markers of dendritic cells cultivated in the presence of EVs indicated that their content disrupts the maturation process. Additionally, microRNAs known to interfere with dendritic cell function, and detected in EVs, matched microRNAs from squamous cell carcinoma patients’ plasma: miR-17-5p in oropharyngeal squamous cell carcinoma, miR-21 in oral squamous cell carcinoma, miR-16, miR-24, and miR-181a circulating in both oral and oropharyngeal squamous cell carcinoma, and miR-23b, which has not been previously described in plasma of head and neck squamous cell carcinoma, was found in plasma from patients with these cancer subtypes. This study contributes with insights on EVs in signaling between cancer and immune cells in squamous cell carcinoma of the head and neck.

Objectives The aim of this study was to evaluate the histomorphologic presentation and the expression of stem cell–related markers in a series of oral solitary fibrous tumors (SFTs). Study Design Histopathological variables and the expression of the standard stem cell markers CD34 and CD99, used for SFT diagnosis, as well as STAT6 were evaluated in 13 oral SFTs. The expression of the cancer stem cell markers CD44, ALDH1, Bmi-1, and Nanog and the tumor suppressor gene p16Ink4a were also investigated. Results The majority of oral SFTs were circumscribed and characterized by a proliferation of spindle cells arranged in a hyalinized stroma. Only 2 oral SFTs showed >4 mitoses/10 high-power fields. Hypercellularity as well as nuclear and cellular pleomorphism were classified as low and moderate in most of the oral SFTs. All oral SFTs were positive for CD34, STAT6, CD44, ALDH1, Bmi-1, and p16Ink4a. CD99 and Nanog expression was observed in 11 and 10 oral SFT cases, respectively. Conclusion We suggest that STAT6 and ALDH1 have relevant diagnostic value. The expression of CD44, ALDH1, Bmi-1, and Nanog, which is observed in cancer stem cells, may confer advantages to oral SFT cells.

To investigate the expression of upstream and downstream targets of mTOR signalling pathway in the secretory carcinoma of salivary gland origin (SCsg). Seven cases of secretory carcinoma diagnosed by a combination of immunohistochemistry and/or molecular testing were retrieved from our pathology files. For comparison purposes, 27 other salivary carcinomas were selected. Immunohistochemical staining was performed against phospho-Akt, PTEN, phospho-mTOR, phospho-4E-BP, eIF4E and phospho-S6 ribosomal protein. With the exception of Akt, all the other proteins were present at some level in the SCsg and in other salivary carcinomas. PTEN was diffusely expressed in 57.1% of SCsg, but only in 14.8% of other salivary carcinomas. mTOR is expressed in more than half of the cases both for SCsg and other salivary tumour types. Most cases of SCsg showed negative expression for S6 ribosomal protein (71.4%) and 4E-BP1 (57.1%). For both groups evaluated, eIF4E was the most expressed protein. SCsg shows different expression patterns for the mTOR signalling molecules, but only eIF4E was highly expressed. This may suggest alternative signalling pathways other than Akt and mTOR in this group of tumours.

OBJECTIVE This study investigated components of the Hedgehog (HH) signaling pathway (SHH, GLI1), cyclin D1, and smooth muscle actin (SMA) in central giant cell granulomas (CGCG). The relationship between these proteins and myofibroblasts was also studied. MATERIAL AND METHODS Twelve cases of non-aggressive CGCG and 11 cases of aggressive CGCG were studied using immunohistochemistry for SHH, GLI1, Cyclin D1, and SMA. RESULTS Associations between all proteins in non-aggressive and aggressive CGCG were not significant (P > .05). All cases of CGCG showed significantly higher expression of SMA compared with the other proteins (P

Background Most patients with head and neck cancer receive nonsteroidal anti-inflammatory drugs concomitant with oncogenic treatment in order to control cardiovascular diseases and chronic inflammatory processes. Inflammation is closely related to neoplastic development and the release of inflammatory cytokines and chemokines represents a crucial event in this relationship. The aim of the present study was to evaluate the effect of acetylsalicylic acid (ASA) and celecoxib treatment in the gene expression pattern of cytokines and chemokines in squamous cell carcinoma (OSCC) cell lines. Materials and methods Cells were treated with plasmatic concentrations of ASA and celecoxib and were submitted to cell viability assay and immunoenzymatic assay to investigate interleukin 6 (IL6) production. Treated cells were collected and a gene expression array was performed using the reverse transcriptase-quantitative polymerase chain reaction. Results Both treatments provoked a discrete inhibitory effect on cell viability and modulated IL6 production. The mRNA expression of several cytokines, chemokines, chemokine receptors, and other chemotaxis-related genes were modulated after treatment with ASA and celecoxib. Conclusion Plasmatic doses of ASA and celecoxib altered the expression of IL6 and the gene expression of chemokines (ligands and receptors) and cytokines in a dose- and time-dependent manner.

Objectives Investigate the DNA copy number and the methylation profile of the homeobox genes HOXA5, HOXA7, HOXA9, HOXB5, HOXB13, HOXC12, HOXC13, HOXD10, HOXD11, IRX4 and ZHX1, and correlate them with clinicopathological parameters and overall survival. Material and methods DNA from OSCC samples and surgical margins were submitted to DNA amplification by qPCR and to DNA methylation analysis using a DNA Methylation PCR Array System. Results HOXA5, HOXB5 and HOXD10 were amplified in surgical margins while HOXA9, HOXB13 and IRX4 were amplified in OSCC. HOXD10 demonstrated hypermethylation in half of the tumor while ZHX1 did not show hypermethylation. No correlation of DNA copy number or methylation with clinicopathological parameters or survival was observed. Conclusion HOXA9, HOXB13 and IRX4 genes appears to be regulated by amplification and HOXD10 by methylation in OSCC. Further studies are needed to determine the role of these events in OSCC development.

Oral squamous cell carcinoma (OSCC) is the most prevalent neoplasia of oral cavity worldwide and prognosis remains unchanged in decades. Recently, different authors reported that head and neck squamous cell carcinomas have a subpopulation of tumor initiating cells that apparently correspond to cancer stem cells (CSC) and are also responsible for tumor growth and metastasis. The purpose of the present study was to investigate the microscopic and phenotypic characteristics of OSCC tumors induced after orthotopic xenoimplantation of SCC9WT cell line and CSC-enriched subpopulation isolated from SCC9 cell line based on high expression of the putative CSC marker CD44. Different numbers of FACS-sorted SCC9 CD44high and CD44low cells as well as SCC9WT (wild type) were transplanted into the tongue of BALB/C nude (NOD/SCID) mice to evaluate their tumorigenic potential. Sixty days post-induction, tumors were morphologically characterized and immunostained for CSC markers (CD44, Nanog and Bmi-1), epithelial-mesenchymal transition (Snail, Slug) and epithelial differentiating cell markers (cytokeratins 4, 13, 15, 17 and 19), as well as E-cadherin and β-catenin. The data presented here shows that SCC9 CD44high cells have higher ability to form tumors than SCC9 CD44low cells, even when significantly lower numbers of SCC9 CD44high cells were transplanted. Immunoassessment of tumors derived from SCC9 CD44high cells revealed high expression of cytokeratin CK19, β-catenin, E-cadherin and CD44, and negative or low expression of CK17, CK4, CK15, CK13, Nanog, Bmi-1, Snail and Slug. While tumors derived from SCC9WT showed high expression of CK17, CK19, CD44, Nanog, Bmi-1, Snail and Slug, and negative or low expression of CK4, CK15, CK13, β-catenin and E-cadherin. Thus, SCC9 CD44high cells were highly tumorigenic, capable of originating heterogeneous tumors and these tumors have a immunohistochemical profile different from those formed by the wild type cell line.

Made available in DSpace on 2018-12-11T17:23:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Objective The aim of this study was to evaluate the effect of chronic smoking on the expression profile of the repair genes MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers and never smokers. Materials and methods The sample consisted of thirty exfoliative cytology smears per group obtained from Smokers and Never Smokers. Total RNA was extracted and expression of the MLH1, MSH2 and ATM genes were evaluated by quantitative real-time and immunocytochemistry. The gene and protein expression data were correlated to the clinical data. Gene expression was analyzed statistically using the Student t-test and Pearson's correlation coefficient, with p

Objective: This study aimed to show schoolchildren the variety of microorganisms present before and after hand hygiene, and to make them aware of the transmission and autoinoculation of pathogenic microorganisms. Study design: This study was approved by ethical committee and is part of the "Projeto Experimentando Ciencia." A total of 27 volunteers from 5 different public schools, aged from 11 to 15 years, participated in this study. A microbiota sample was collected using sterile swab and Sabouraud dextrose agar plates before and after hand hygiene orientation (World Health Organization [WHO] technique 1). After 1 week, plates were shown and explained to the participating children. Different materials were used for hand hygiene in each school. Results: A total of 35% used water and paper towels to sanitize the hands; 32% used water, soap and paper towels; 30% used only water; and 3% used soap and water. A larger variety and number of colonies were found before sanitization than after, even in the group that washed hands only with water. Fungi such as Penicillium, Aspergillus, Cladosporium, and nonsporulated types, as well as bacteria, were identified on the samples. One week after the procedure, teachers reported children changed their hand hygiene habits. Conclusion: Visualization of fungi and bacteria culture improved schoolchildren hand hygiene.

In response to stimuli in the microenvironment, macrophages adopt either the M1 or M2 phenotype to coordinate the tissue repair process. Photobiomodulation (PBM) plays an important role in the modulation of acute inflammation, including cellular influx, macrophage polarization, and the release of inflammatory mediators. The aim of the present study was to evaluate the effects of red and infrared PBM on the mRNA expression of cytokines and chemokines in macrophages polarized to the M1 and M2 phenotypes. J774 macrophages activated to induce M1 (lipopolysaccharide + interferon gamma) or M2 (interleukin-4) phenotypes were irradiated with red or infrared PBM (1 J). After 4 and 24 h, gene expression was analyzed by qPCR. PBM at 660 nm decreased the mRNA expression of CCL3, CXCL2, and TNF-α in M1 macrophages and CXCL2 in M2 macrophages 4 h after irradiation. Similarly, PBM at 780 nm decreased mRNA expression levels of CCL3 and IL-6 by M1 macrophages 24 h after irradiation. Moreover, PBM at 780 nm increased the mRNA expression of TGFβ1 4 h after irradiation and decreased the expression of this gene after 24 h in M2 macrophages. Although red and infrared PBM were able to modulate and reduce M1/M2a-related markers, infrared laser irradiation promoted a temporal increase in the expression of TGFβ1 in M2 macrophages. Thus, depending on the time PBM is used on injured tissue, different parameters can promote optimal results by modulating specific macrophage phenotypes.

Background Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy and is successfully treated by surgery and radiation. However, some patients have recurrent tumours and in these cases, few treatments options are available. Cancer stem cells (CSC) have been observed and isolated from different solid tumours based on the expression of stem cell markers. These cells are associated with tumour initiation, progression as well as treatment resistance. In this study, the expression of stem cell markers CD44, Bmi1, Oct4 and Nanog was evaluated in non-neoplastic salivary tissue and in MEC. Methods Twenty-eight samples of MEC and their corresponding non-neoplastic salivary tissue were examined by immunohistochemistry and the stem cell markers expression was correlated with histological and clinical parameters. Results CD44 was expressed in the membrane of serous and mucous acini as well as in the ductal cells in normal gland tissue. Bmi1, Oct4 and Nanog were mainly expressed in ductal structures. In MEC, CD44 and Bmi1 showed strong expression in all types of neoplastic cells and both markers revealed intense expression in tumour invasive front. Oct4 and Nanog protein expression was associated with desmoplasia and perineural invasion. Only Oct4 positive tumours were associated with dissociative growth pattern and committed margins. Conclusion The stem cell markers CD44, Bmi1, Oct4 and Nanog are frequently expressed in MEC in relation to normal salivary gland and Oct4 and Nanog expression may contribute to aggressiveness and worst prognosis in MEC patients.

Objective: This study investigated components of the HH pathway (SHH, GLI1, and cyclin D1) in central giant cell granulomas (CGCG). The relationship between these proteins and myofibroblasts was also studied. Material and Methods: Twelve cases of non-aggressive CGCG and 11 cases of aggressive CGCG were studied using immunohistochemistry for SHH, GLI1, Cyclin D1, and SMA. Results: Associations between all proteins in non-aggressive and aggressive CGCG were not significant (P > .05). All cases of CGCG showed significantly higher expression of SMA compared to the other proteins (P

Receptor-interacting proteins are a family of serine/threonine kinases, which integrate extra and intracellular stress signals caused by different factors, including infections, inflammation and DNA damage. Receptor-interacting serine/threonine-protein kinase 2 (RIP-2) is a member of this family and an important component of the nuclear factor NF-kappa-B signaling pathway. The corresponding human gene RIPK2 generates two transcripts by alternative splicing, the full-length and a short transcript. The short transcript has a truncated 5’ sequence, which results in a predicted isoform with a partial kinase domain but able to transduce signals through its caspase recruitment domain. In this study, the expression of RIPK2 was investigated in human tissue samples and, in order to determine if both transcripts are similarly regulated at the transcriptional level, cancer cell lines were submitted to temperature and acid stresses. We observed that both transcripts are expressed in all tissues analyzed, with higher expression of the short one in tumor samples, and they are differentially regulated following temperature stress. Despite transcription, no corresponding protein for the short transcript was detected in tissues and cell lines analyzed. We propose that the shorter transcript is a noncoding RNA and that its presence in the cell may play regulatory roles and affect inflammation and other biological processes related to the kinase activity of RIP-2., Instituto de Biotecnologia y Biologia Molecular

Oral squamous cell carcinoma (OSCC) is an extremely aggressive disease associated with a poor prognosis. Previous studies have established that cancer stem cells (CSCs) actively participate in OSCC development, progression and resistance to conventional treatments. Furthermore, CSCs frequently exhibit a deregulated expression of normal stem cell signalling pathways, thereby acquiring their distinctive abilities, of which self-renewal is an example. In this study, we examined the effects of GLI3 knockdown in OSCC, as well as the differentially expressed genes in CSC-like cells (CSCLCs) expressing high (CD44high) or low (CD44low) levels of CD44. The prognostic value of GLI3 in OSCC was also evaluated. The OSCC cell lines were sorted based on CD44 expression; gene expression was evaluated using a PCR array. Following this, we examined the effects of GLI3 knockdown on CD44 and ESA expression, colony and sphere formation capability, stem-related gene expression, proliferation and invasion. The overexpression of genes related to the Notch, transforming growth factor (TGF)β, FGF, Hedgehog, Wnt and pluripotency maintenance pathways was observed in the CD44high cells. GLI3 knockdown was associated with a significant decrease in different CSCLC fractions, spheres and colonies in addition to the downregulation of the CD44, Octamer-binding transcription factor 4 (OCT4; also known as POU5F1) and BMI1 genes. This downregulation was accompanied by an increase in the expression of the Involucrin (IVL) and S100A9 genes. Cellular proliferation and invasion were inhibited following GLI3 knockdown. In OSCC samples, a high GLI3 expression was associated with tumour size but not with prognosis. On the whole, the findings of this study demonstrate for the first time, at least to the best of our knowledge, that GLI3 contributes to OSCC stemness and malignant behaviour. These findings suggest the potential for the development of novel therapies, either in isolation or in combination with other drugs, based on CSCs in OSCC.

Background Tongue squamous cell carcinoma (SCC) contains a cell subpopulation referred to as cancer stem cells (CSCs), which are responsible for tumor growth, metastasis, and resistance to chemotherapy and radiotherapy. The CSC markers have been used to isolate these cells and as biomarkers to predict overall survival. Methods The CSC markers CD44, NANOG, OCT4, and BMI1 were investigated using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry and correlated with clinicopathological parameters. Results The CD44 overexpression was associated with disease-related death (P = 0.02) and worst prognosis. NANOG was upregulated in nontumoral margins and associated with T1/T2 classification, lymph node metastasis, and worst prognosis. OCT4 was associated with lymph node metastasis and worst overall survival. BMI1 and CD44v3 were overexpressed in tongue SCC. Coexpression of CD44++ /NANOG++ was associated with worst overall survival when compared with patients with CD44-/+ /NANOG-/+ . Conclusion The CSC markers might play an important role not only in CSC trait acquisition but also in tongue SCC development and progression.

Low-level laser irradiation (LLLI) is increasingly used to treat musculoskeletal disorders, with satisfactory results described in the literature. Skeletal muscle satellite cells play a key role in muscle regeneration. The aim of the present study was to evaluate the effect of LLLI on cell viability, creatine kinase (CK) activity, and the expression of myogenic regulatory factors in C2C12 myoblasts during the differentiation process. C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum and submitted to irradiation with GaAlAs diode laser (wavelength, 780 nm; output power, 10 mW; energy density, 5 J/cm2). Cell viability and the expression of myogenic regulatory factors were assessed 24, 48, and 72 h after irradiation by 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. CK activity was analyzed at 24 and 72 h. An increase in cell viability was found in the laser group in comparison to the control group at all evaluation times. CK activity was significantly increased in the laser group at 72 h. Myogenin messenger RNA (mRNA) demonstrated a tendency toward an increase in the laser group, but the difference in comparison to the control group was non-significant. In conclusion, LLLI was able to modulate cell viability and CK activity in C2C12 myoblasts during the differentiation process.

Background Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyses the expression of DNMTs in odontogenic tumours. Methods Formalin-fixed and paraffin-embedded tissue samples of 20 ameloblastomas, 10 calcifying cystic odontogenic tumours, 10 calcifying epithelial tumours, 10 adenomatoid odontogenic tumours, 10 keratocystic odontogenic tumours, five ameloblastic fibromas, two ameloblastic fibro-odontomas, four central odontogenic fibromas, seven peripheral odontogenic fibromas and 10 odontogenic myxomas were included. Immunohistochemical expression of DNMT1, 3A and 3B was assessed using a semi-quantitative analysis, and also a correlation with p21, p27 and E-cadherin immunoexpression was made. Results DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. DNMT1 expression was directly correlated with p27 expression in ameloblastomas. Conclusion The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Objective Oral non-Hodgkin lymphomas (NHLs) are an extensive group of malignant lymphoid cell neoplasms that are the second most common group of oral cancers. Subtyping NHL is important to plan for appropriate treatment, and the analysis of clonality is in many instances used for helping in the diagnosis of NHL. Thus, the aim of this study was to analyze immunoglobulin heavy chain (IgH) gene rearrangement in a series of oral B cell lymphomas to investigate the sensitivity of seminested polymerase chain reaction (snPCR). Study Design Paraffin embedded tissue samples from 16 cases of oral B cell lymphomas were retrieved and subjected to snPCR to investigate the IgH gene rearrangement. Results The results showed monoclonal IgH rearrangement in 85.7% of the cases studied, as represented by finding one band within the expected range of amplification. Conclusions This study found that snPCR is a consistent method for the detection of gene rearrangement in paraffin-embedded tissue.

Oral manifestations of common variable immunodeficiency (CVID) are rare, have rarely been studied and have given controversial results. There are few data about IgA, IgG, and IgM antibody salivary levels in the literature, and there are few papers about the clinical impact of antibody deficiencies and CVID on the oral health of such patients. The aim of this study was to measure serum and salivary IgA, IgG, and IgM levels in CVID participants and controls, and to associate immunoglobulin levels with caries and periodontal disease. This was a case-control study involving 51 CVID individuals and 50 healthy controls. All participants underwent examination for dental caries and periodontal disease. Blood and whole saliva samples were collected on the same day of the oral examination. Serum IgA, IgM, and IgG levels were measured by turbidimetry and salivary IgA, IgM, and IgG titers were assessed by enzyme-linked immunosorbent assay. Incidences of caries and gingivitis were significantly higher in the CVID group than in the control group (p0.05 for both). Although CVID was associated with increased susceptibility to caries and gingivitis, it was not associated with low salivary levels of IgA and IgM. Resumo As manifestações orais em pacientes com imunodeficiência comum variável (ICV) têm sido pouco estudadas e com resultados variados. Há escassos dados na literatura sobre os níveis de IgA, IgG e IgM na saliva, e pouco se sabe sobre o impacto clínico da deficiência destes anticorpos sobre a saúde bucal de pacientes com ICV. O objetivo deste estudo foi medir os níveis séricos e salivares de IgA, IgG e IgM em indivíduos com ICV e controles, e associar os níveis de imunoglobulinas com cárie e doença periodontal. Este foi um estudo de caso-controle, envolvendo 51 indivíduos ICV e 50 controles saudáveis. Todos os participantes foram examinados para cárie e doença periodontal. As amostras de sangue e saliva foram coletadas no mesmo dia do exame intraoral. Os níveis de IgA, IgM e IgG foram medidos por turbidimetria, e os títulos salivares de IgA, IgM e IgG foram avaliados através método imunoenzimático (ELISA). As incidências de cáries e gengivite foram significativamente maiores no grupo ICV do que no grupo controle (p0,05 para ambos). Embora ICV foi associado com um aumento da susceptibilidade à cárie e gengivite, não estava associado com baixos níveis salivares de IgA e IgM.

We conducted a genome-wide association study of oral cavity and pharyngeal cancer in 6,034 cases and 6,585 controls from Europe, North America and South America. We detected eight significantly associated loci (P < 5 × 10(-8)), seven of which are new for these cancer sites. Oral and pharyngeal cancers combined were associated with loci at 6p21.32 (rs3828805, HLA-DQB1), 10q26.13 (rs201982221, LHPP) and 11p15.4 (rs1453414, OR52N2-TRIM5). Oral cancer was associated with two new regions, 2p23.3 (rs6547741, GPN1) and 9q34.12 (rs928674, LAMC3), and with known cancer-related loci-9p21.3 (rs8181047, CDKN2B-AS1) and 5p15.33 (rs10462706, CLPTM1L). Oropharyngeal cancer associations were limited to the human leukocyte antigen (HLA) region, and classical HLA allele imputation showed a protective association with the class II haplotype HLA-DRB1*1301-HLA-DQA1*0103-HLA-DQB1*0603 (odds ratio (OR) = 0.59, P = 2.7 × 10(-9)). Stratified analyses on a subgroup of oropharyngeal cases with information available on human papillomavirus (HPV) status indicated that this association was considerably stronger in HPV-positive (OR = 0.23, P = 1.6 × 10(-6)) than in HPV-negative (OR = 0.75, P = 0.16) cancers. Genotyping performed at the Center for Inherited Disease Research (CIDR) was funded through the U.S. National Institute of Dental and Craniofacial Research (NIDCR) grant 1X01HG007780-0. Genotyping for shared controls with the Lung OncoArray initiative was funded through the grant X01HG007492-0. Corina Lesseur undertook this work during the tenure of a Postdoctoral Fellowship awarded by the International Agency for Research on Cancer. The funders did not participate in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We acknowledge all of the participants involved in this research and the funders and support. We thank Dr. Leticia Fernandez (Instituto Nacional de Oncologia y Radiobiologia, La Habana, Cuba) for her contribution to the IARC ORC multicenter study. We are also grateful to Sergio Koifman (Escola Nacional de Saúde Pública, Rio de Janeiro, Brazil) for his contribution to the IARC Latin America multicenter study (Sergio Koifman passed away in May 2014) and to Xavier Castellsagué from the ARCAGE Barcelona Center who recently passed away (June 2016). The University of Pittsburgh head and neck cancer case-control study is supported by National Institutes of Health grants P50 CA097190 and P30 CA047904. The Carolina Head and Neck Cancer Study (CHANCE) was supported by the National Cancer Institute (R01-CA90731). The Head and Neck Genome Project (GENCAPO) was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant numbers 04/12054-9 and 10/51168-0). The authors thank all the members of the GENCAPO team. The HN5000 study was funded by the National Institute for Health Research (NIHR) under its Programme Grants for Applied Research scheme (RP-PG-0707-10034), the views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The Toronto study was funded by the Canadian Cancer Society Research Institute (020214) and the National Cancer Institute (U19 CA148127) and the Cancer Care Ontario Research Chair. The alcohol-related cancers and genetic susceptibility study in Europe (ARCAGE) was funded by the European Commission’s 5th Framework Program (QLK1-2001-00182), the Italian Association for Cancer Research, Compagnia di San Paolo/FIRMS, Region Piemonte, and Padova University (CPDA057222).The Rome Study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 2011 10491 and IG2013 14220 to SB, and Fondazione Veronesi to SB. The IARC Latin American study was funded by the European Commission INCO-DC programme (IC18-CT97-0222), with additional funding from Fondo para la Investigacion Cientifica y Tecnologica (Argentina) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (01/01768-2). The IARC Central Europe study was supported by European Commission’s INCO-COPERNICUS Program (IC15-CT98-0332), NIH/National Cancer Institute grant CA92039, and the World Cancer Research Foundation grant WCRF 99A28.The IARC Oral Cancer Multicenter study was funded by: grant S06 96 202489 05F02 from Europe against Cancer; Grants FIS 97/0024, FIS 97/0662, and BAE 01/5013 from Fondo de Investigaciones Sanitarias, Spain; UICC Yamagiwa-Yoshida Memorial International Cancer Study; National Cancer Institute of Canada; Italian Association for Research on Cancer; and the Pan American Health Organization. The coordination of EPIC study is financially supported

Approximately 300 homeobox loci were identified in the euchromatic regions of the human genome, of which 235 are probable functional genes and 65 are likely pseudogenes. Many of these genes play important roles in embryonic development and cell differentiation. Dysregulation of homeobox gene expression is a frequent occurrence in cancer. Accumulating evidence suggests that as genetics disorders, epigenetic modifications alter the expression of oncogenes and tumor suppressor genes driving tumorigenesis and perhaps play a more central role in the evolution and progression of this disease. Here, we described the current knowledge regarding homeobox gene DNA methylation in human cancer and describe its relevance in the diagnosis, therapeutic response and prognosis of different types of human cancers.

Made available in DSpace on 2019-09-12T16:57:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2012 Objective. The purposes of this study were to assess clinical, histopathological and immunohistochemical features of 22 oral neurofibromas (NFs) and discuss with previously described literature, addressing the main aspects regarding the differential diagnosis. Materials and methods. Immunohistochemical reactions included S-100, CD34, GLUT-1, EMA, Ki-67, p53 and Collagen IV and histochemical reactions for Alcian blue. Results. Clinically, the preferential location was the maxillary bones, tongue and buccal mucosa. Microscopically, widely spread spindle-shaped cells with scant cytoplasm and elongated nuclei were observed. Immunostaining revealed that the tumor cells weakly expressed GLUT-1, Collagen IV, Ki-67 and p53. They were variably positive for CD34, S-100 protein and membrane epithelial antigen (EMA). Conclusions. The different types of nerve sheath cells observed in the present series reinforce the presence of heterogeneous population in NFs. The strong positivity for S-100 suggests that the lesions were more composed by S-100-positive Schwann cells than other cells. Besides, the high number of CD34-positive cells suggests that this marker can be useful for the differential diagnosis of NFs against PEN, traumatic neuromas and Schwannomas. Finally, the low immunostaining for p53 and Ki-67 may indicate that NFs massively composed by S-100-positive Schwann cells present low potential of aggressiveness and malignant transformation. [Campos, Marcia Sampaio] Universidade de Taubaté (Unitau), Dept Dent Oral Pathol Oral Med, BR-12020330 Taubate, SP, Brazil [Fontes, Alexandra; Marocchio, Luciana Sassa; Nunes, Fabio Daumas; Orsini Machado de Sousa, Suzana Cantanhede] Univ Sao Paulo, Sch Dent, Dept Stomatol Oral Pathol, Sao Paulo, Brazil

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:26:26Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:26:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-04-01 The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC. Department of Oral Pathology School of Dentistry University of São Paulo, Av. Prof. Lineu Prestes 2227, Cidade Universitária, 05508-000 São Paulo Department of Head and Neck Surgery School of Medicine University of São Paulo, Av. Dr. Arnaldo 455, Cerqueira César, 01246903 São Paulo Head and Neck Surgery Division Heliópolis Hospital Complex, Rua Cônego Xavier 276, Sacomã, 04231-030 São Paulo Department of Head and Neck Surgery Arnaldo Vieira de Carvalho Cancer Institute, Rua Dr Cesário Motta Junior 112, Vila Buarque, 01221020 São Paulo Department of Molecular Biology São José do Rio Preto School of Medicine, Av. Brigadeiro Faria Lima 5416-Vila São Pedro, 15090-000 São José do Rio Preto Department of Genetics and Evolutionary Biology Institute of Biosciences University of São Paulo, R. do Matão, travessa 14 321, Cidade Universitária, 05508-090 São Paulo Department of Genetics Biosciences Institute University of São Paulo, Rua do Matão, trav. 14 321, Cidade Universitária, 05508-090 São Paulo School of Medicine USP, São Paulo Albert Einstein Jewish Teaching and Research Institute (IIEP), São Paulo School of Medicine of São José do Rio Preto São José Dos Campos Dental School UNESP Cancer Institute Arnaldo Vieira de Carvalho, São Paulo School of Dentistry USP, São Paulo Heliópolis Hospital, São Paulo School of Medicine of Ribeirão Preto USP School of Public Health USP, São Paulo University of Vale do Paraíba, São José dos Campos School of Medicine UNIFESP, São Paulo Faculty of Pharmaceutical Sciences of Ribeirão Preto USP, SP São José Dos Campos Dental School UNESP

The purpose of the present study is to verify a possible association between herpesviruses and periodontal pathogens in individuals with human immunodeficiency virus (HIV) and periodontitis.Twenty-seven patients with HIV and chronic periodontitis and 23 patients with HIV and gingivitis were included in the study. Probing depth, clinical attachment loss, gingival index, and plaque index were recorded. Blood, saliva, and subgingival plaque were processed for viral and bacterial identification. Bacteria were identified by 16S rRNA-based polymerase chain reaction and viruses by the nested polymerase chain reaction.For the chronic periodontitis group, Epstein-Barr (EBV)-1 (70.4%) and Tannerella forsythia (Tf) (51.8%) presented higher detection in subgingival plaque and saliva (81.5% and 40.7%, respectively) than in blood (22% and 0%, respectively) (P0.005 and P0.0001, respectively). Porphyromonas gingivalis (Pg) was more frequent in subgingival plaque (77.7%; P0.0001). In the gingivitis group, Pg and human cytomegalovirus (HCMV) presented higher frequency in subgingival plaque (95.6% and 91.3%, respectively; P0.0001 and P = 0.004). Tf and EBV-1 were detected more frequently in subgingival plaque (47.8% and 78.3%, respectively) and saliva (52.2% and 52.2%, respectively; P = 0.004 and P0.005) than in blood. EBV-1, EBV-1-HCMV, and presence of different viruses presented an association with periodontitis in saliva.No association was detected for herpesviruses and periodontal pathogens in patients who are HIV-positive with periodontitis. EBV-1 and coinfection (EBV-1-HCMV) were associated with patients who are HIV-positive with periodontitis.

J Oral Pathol Med (2012) 41: 80–85 Background: Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, viral, fungal or host tissues. They are responsible for promoting the production of cytokines and chemokines, increasing the expression of costimulatory molecules and influencing the T Helper response (Th) toward either a Th1 or Th2 profile, thereby modulating the regulatory T cell response and controlling the integrity of the epithelial barrier. The key factors responsible for increased susceptibility to recurrent aphthous ulceration (RAU) are unclear, and because TLRs are involved in both immune regulation and control of the epithelial barrier, a deficiency in TLR activity is likely to cause increased susceptibility. Methods: We investigated the gene expression of TLRs one through 10 in tissue samples and peripheral blood mononuclear cells (PBMC) of RAU patients in comparison to healthy controls using real-time quantitative reverse transcription PCR. Results: The analysis of mRNA expression levels in oral lesion showed significant (P

Objective Variations in genes that are critical for tooth formation may contribute to the tooth agenesis. MMPs are potential candidate genes for dental alterations based on the roles they play during embryogenesis. The aim of this study was to investigate the possible association between MMP1, MMP3, and MMP20 and tooth agenesis. Methods One hundred sixty-seven nuclear families from two different populations were analysed, 116 from Brazil and 51 from Turkey. Probands had at least one congenitally missing tooth. DNA samples were obtained from blood or saliva samples and genotyping was performed using TaqMan chemistry. In addition, Mmp20 was selected for quantitative real-time polymerase chain reaction analysis with SYBR Green I Dye in mouse tooth development. Results Associations between tooth agenesis and MMP1 (p = 0.007), and MMP20 (p = 0.03) were found in Brazilian families. In the total dataset, MMP20 continued to be associated with tooth agenesis (p = 0.01). Mmp20 was not expressed during the initial stages of tooth development. Conclusion Our findings provide evidence that MMP1 and MMP20 play a role in human tooth agenesis.

Aims: To analyse the expression of three homeoboxgenes (HOXA7, PITX1 and PRRX1) in oral squanouscell carcinomas (OSCC) and the relationship of suchexpression to certain distinct histopathological featuresof OSCC and in comparison to adjacent non-neoplasticepithelium (NT).Methods and results: Digoxigenin-labelled riboprobesthat are specific for each homeobox gene were gener-ated and in situ hybridization was carried out on frozensections. In NT samples, HOXA7 and PITX1 transcriptswere found more frequently in all epithelial layers,while PRRX1 was expressed in the basal layer. WithOSCC samples, expression of the three genes wasassociated with all histological features. However,the HOXA7 and PITX1 signals were more intense insheets and nests and PRRX1 in small nests and isolatedcells.Conclusion: HOXA7, PIXT1 and PRRX1 homeoboxgenes have different patterns of expression in OSCCdepending on its histological features.Keywords: homeobox genes, oral squamous cell carcinomaAbbreviations: AS, antisense; ISH, in situ hybridization; NT, adjacent non-tumoral oral epithelium; OSCC, oralsquamous cell carcinoma; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; S, sense;SDS, sodium dodecyl sulphate

In this study the presence of periodontopathic pathogens in atheromatous plaques removed from coronary arteries of patients with chronic periodontitis and periodontally healthy subjects by PCR was detected. Our results indicate a significant association between the presence of Porphyromonas gingivalis and atheromas, and the periodontal bacteria in oral biofilm may find a way to reach arteries.

Previous studies indicated that patients with atherosclerosis are predominantly infected by human cytomegalovirus (HCMV), but rarely infected by type 1 Epstein-Barr virus (EBV-1). In this study, atheromas of 30 patients who underwent aortocoronary bypass surgery with coronary endartherectomy were tested for the presence of these two viruses. HCMV occurred in 93.3% of the samples and EBV-1 was present in 50% of them. Concurrent presence of both pathogens was detected in 43.3% of the samples.

In this study, Bmp-4, Wnt-5a and Shh gene expressions were compared during early craniofacial development in mice by comparative non-isotopic in situ hybridization. Wild-type C57BL/6J mice were studied at various stages of embryonic development (from 8.5- to 13.5-day-old embryos--E8.5-13.5). During early odontogenesis, transcripts for Bmp-4, Shh and Wnt-5a were co-localised at the tooth initiation stage. At E8.5, Shh mRNA expression was restricted to diencephalon and pharyngeal endoderm. Before maxillae and mandible ossification, Bmp-4 and Wnt-5a signals were detected in the mesenchymal cells and around Meckel's cartilage. During palatogenesis, Shh was expressed only in the epithelium and Wnt-5a only in the mesenchyme of the elevating palatal shelves. During tongue development, Shh expression was found in mesenchyme, probably contributing to tongue miogenesis, while Wnt-5a signal was in the epithelium, possibly during placode development and papillae formation. Taken together, these findings suggest that Bmp-4, Shh and Wnt-5a gene expressions may act together on the epithelial-mesenchymal interactions occurring in several aspects of the early mouse craniofacial development, such as odontogenesis, neuronal development, maxillae and mandible ossification, palatogenesis and tongue formation.

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.

Background: Oncogenic Wnt/β-catenin signaling occurs in numerous types of cancers, but little is known about the role of the Wnt protein family member, WNT-5A, in lip carcinogenesis. The aim of this study was to investigate WNT-5A, β-catenin, and matrix metalloproteinase (MMP)-3 protein expression in actinic cheilitis (AC), and lip squamous cell carcinoma (LSCC). Methods: Twenty-one cases of AC, and fifty-one cases of LSCC were analyzed, with normal lip mucosa used as a control. Qualitative and semi-quantitative analyses of WNT-5A, β-catenin, and MMP-3 immunostaining pattern and cellular distribution were performed. Results: WNT-5A was observed in more than 50% of the cells, scattered in all layers of AC, in contrast to the absence of immunostaining in normal lip mucosa. AC presented a higher level of WNT-5A expression than LSCC (P = 0.0289, Fisher test), while MMP-3 immunoexpression was statistically more significant in LSCC than in AC (P = 0.0285, Fisher test). Immunolabeling of β-catenin protein was differentially distributed between samples; the majority of AC cases (61.90%) demonstrated a membranous-cytoplasmic pattern, while a considerable number of LSCC cases (29.41%) revealed a cytoplasmic pattern, instead of the usual membranous pattern. Conclusions: The present results suggest that WNT-5A may be an important marker during initial events of AC malignant transformation, in which non-canonical and canonical Wnt/β-catenin signaling pathways could be involved. Additionally, WNT-5A might recruit other events in LSCC, such as MMP-3 protein synthesis, as its presence is increased in established malignant processes without β-catenin dependency.

Herpesviruses may be related to the etiology of aggressive periodontitis (AgP) and chronic periodontitis (CP) by triggering periodontal destruction or by increasing the risk for bacterial infection. This case-control study evaluated the presence of herpes simplex virus type I (HSV-1), Epstein-Barr virus type I (EBV-1), human cytomegalovirus (HCMV), Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia (previously T. forsythensis) in patients with generalized AgP (AgP group), CP (CP group), or gingivitis (G group) and in healthy individuals (C group).Subgingival plaque samples were collected with paper points from 30 patients in each group. The nested polymerase chain reaction (PCR) method was used to detect HSV-1, EBV-1, and HCMV. Bacteria were identified by 16S rRNA-based PCR.HSV-1, HCMV, and EBV-1 were detected in 86.7%, 46.7%, and 33.3% of the AgP group, respectively; in 40.0%, 50.0%, and 46.7% of the CP group, respectively; in 53.3%, 40.0%, and 20.0% of the G group, respectively; and in 20.0%, 56.7%, and 0.0% of the C group, respectively. A. actinomycetemcomitans was detected significantly more often in the AgP group compared to the other groups (P0.005). P. gingivalis and T. forsythia were identified more frequently in AgP and CP groups, and AgP, CP, and G groups had higher frequencies of P. intermedia compared to the C group.In Brazilian patients, HSV-1 and EBV-1, rather than HCMV, were more frequently associated with CP and AgP.

Introduction: It is still controversial whether human papillomavirus (HPV) can be considered a risk factor in oral carcinogenesis. The aim of this study was to detect HPV DNA in 50 cases diagnosed as oral leukoplakias, with different degrees of epithelial dysplasia, and as oral squamous cell carcinomas, using in situ hybridization with signal amplification (CSA-ISH). Methods: HPV DNA was assessed in paraffin sections using CSA-ISH with a wide-spectrum biotinylated DNA probe. In HPV-positive cases, genotyping with specific probes to HPV types 6/11, 16/18 and 31/33 was performed. Results: The overall prevalence of HPV infection was 24%, markedly higher than that found in the control group. Results showed a discrete proportional relationship in the indices found in leukoplakia with no dysplasia, leukoplakia with dysplasia, and squamous cell carcinoma, but this was not statistically significant. When separating the group of leukoplakia by degrees of dysplasia, this relation of proportion was not observed. In genotyping, HPV types 16/18 were the most prevalent, and types 6/11 were only found in groups of mild or no dysplasia. Conclusion: The results suggest that HPV is not likely to play a role in the progression of malignant transformation in oral lesions. Nevertheless, the increased prevalence of HPV infection compared to normal oral mucosa and the fact that high-risk HPV types were the most frequently identified do not allow the exclusion of HPV as a risk factor in oral carcinogenesis.

Objective: To compare the diagnostic accuracy of immunohistochemistry compared to real-time PCR (using a simple phenol-chloroform DNA extraction protocol) in the detection of HHV8 in small oral biopsies of Kaposi sarcoma. Also to validate the use of this DNA extraction protocol to extract HHV8 DNA.Material and methods: Seventeen cases of oral KS were examined. Data including gender, age, and anatomic location were obtained from patient´s records and histological sections stained with hematoxylin and eosin (H&E) were reviewed. Immunohistochemistry was used to detect HHV8 in lesions of interest, as well as real-time PCR.Results: All the patients were HIV positive, the mean age was 35.5 years old, and the affected oral sites were palate (47%), gingiva (29.4%), tongue (11.8%), lip (5.9%), and oral floor (5.9%). Fifteen samples showed positive staining for HHV8 and only two samples were negative. The same results were observed using real-time PCR HHV8-DNA detection.Relevance: Our findings suggest that immunohistochemistry is faster and cheaper to perform than real-time PCR and was shown to have similar levels of sensitivity and accuracy for detection of HHV8 even in small biopsies. Additionally DNA extraction using a non-commercial kit, as done in this study can further reduce the costs to a pathology service.

M1 profile macrophages exert a major influence on initial tissue repair process. Few days after the occurrence of injury, macrophages in the injured region exhibit a M2 profile, attenuate the effects of the M1 population, and stimulate the reconstruction of the damaged tissue. The different effects of macrophages in the healing process suggest that these cells could be the target of therapeutic interventions. Photobiomodulation has been used to accelerate tissue repair, but little is known regarding its effect on macrophages. In the present study, J774 macrophages were activated to simulate the M1 profile and irradiated with two different sets of laser parameters (780 nm, 70 mW, 2.6J/cm(2), 1.5s and 660 nm, 15 mW, 7.5 J/cm(2), 20s). IL-6, TNF-α, iNOS and COX-2 gene and protein expression were analyzed by RT-qPCR and ELISA. Both lasers were able to reduce TNF-α and iNOS expression, and TNF-α and COX-2 production, although the parameters used for 780 nm laser provided an additional decrease. 660 nm laser parameters resulted in an up-regulation of IL-6 expression and production. These findings imply a distinct, time-dependent modulation by the two different sets of laser parameters, suggesting that the best modulation may involve more than one combination of parameters.

This study analyzed the effect of pre-injury and post-injury irradiation with low-level laser therapy (LLLT) on the mRNA expression of myogenic regulatory factors and interleukin 6 (IL-6) during the skeletal muscle repair. Male rats were divided into six groups: control group, sham group, LLLT group, injury group; pre-injury LLLT group, and post-injury LLLT group. LLLT was performed with a diode laser (wavelength 780 nm; output power 40 mW' and total energy 3.2 J). Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly onto the belly of the tibialis anterior (TA) muscle. After euthanasia, the TA muscle was removed for the isolation of total RNA and analysis of MyoD, myogenin, and IL-6 using real-time quantitative PCR. Significant increases were found in the expression of MyoD mRNA at 3 and 7 days as well as the expression of myogenin mRNA at 14 days in the post-injury LLLT group in comparison to injury group. A significant reduction was found in the expression of IL-6 mRNA at 3 and 7 days in the pre-injury LLLT and post-injury LLLT groups. A significant increase in IL-6 mRNA was found at 14 days in the post-injury LLLT group in comparison to the injury group. LLLT administered following muscle injury modulates the mRNA expression of MyoD and myogenin. Moreover, the both forms of LLLT administration were able to modulate the mRNA expression of IL-6 during the muscle repair process.

Most polarized epithelia constrain solute diffusion between luminal and interstitial compartments using tight junctions and generate mechanical strength using adherens junctions. These intercellular junctions are typically portrayed as incongruent macromolecular complexes with distinct protein components. Herein, we delineate the molecular composition and subdomain architecture of an intercellular junction between sensory and non-sensory cells of the inner ear. In this junction, claudins partition into claudin-14 and claudin-9/6 subdomains that are distinguishable by strand morphology, which contrasts with in vitro data that most claudins co-assemble into heteromeric strands. Surprisingly, canonical adherens junction proteins (p120ctn, α- and β-catenins) colocalize with the claudin-9/6 subdomain and recruit a dense cytoskeletal network. We also find that catenins colocalize with claudin-9 and claudin-6, but not claudin-14, in a heterologous system. Together, our data demonstrate that canonical tight junction and adherens junction proteins can be recruited to a single junction in which claudins partition into subdomains and form a novel hybrid tight junction with adherens junction organization.

FUNDAMENTOS: A deleção e mutação do gene CDKN2a que codifica um inibidor específico da ciclina dependente de quinase 4, a proteína p16, têm sido implicadas na tumorigênese do melanoma cutâneo. Entretanto, pouco se conhece sobre essas alterações genéticas em melanomas de mucosa oral. OBJETIVOS: Verificar a presença de alterações no gene p16 e sua expressão protéica em melanomas esporádicos orais e cutâneos. MATERIAL E MÉTODOS: Avaliaram-se 36 espécimes de melanoma primário (sete orais e 29 cutâneos). Analisaram-se três exons do gene p16, pela técnica da reação em cadeia da polimerase/polimorfismo conformacional de fita simples do DNA.Verificou-se a expressão tecidual de proteína p16 por técnica imuno-histoquímica. Relacionaram-se os resultados com a espessura dos melanomas cutâneos. RESULTADOS: Cinco dos sete melanomas orais e 17 dos 29 melanomas cutâneos apresentaram indício de alteração no gene p16. Alterações do exon 2 foram as mais freqüentes, sendo 19 casos nos produtos obtidos com o mesmo iniciador. Observou-se expressão tecidual de p16 em apenas um melanoma oral, em 10/13 (76,9%) casos de melanoma cutâneo de espessura até 1mm e em sete de oito (87,5%) casos de espessura superior a 1mm. CONCLUSÃO: A freqüência de indícios de alteração na análise genética de p16 nos melanomas de mucosa oral foi de 71,42% e de 58,6% nos cutâneos. É possível sugerir a participação de alterações do gene p16 na patogenia do melanoma esporádico de mucosa oral. Não houve relação da sugestão de alteração genética do gene p16 e de sua expressão tecidual com a espessura dos melanomas cutâneos de diferentes subtipos histológicos.

Collagenous fibroma (desmoplastic fibroblastoma) is a rare benign soft tissue tumor with a fibroblastic origin. In oral mucosa only two cases have been described in the literature. We describe the case of a 42-year-old white woman whose complaint was a painless, slow-growing mass under the prosthesis. Histopathologic features included sparsely distributed stellate or spindle fibroblasts within a rich collagenous stroma. Tumor cells were diffusely stained for vimentin and rare cells stained for smooth muscle actin and factor XIIIa. Total surgical excision was performed and no recurrence is expected. O fibroma colagenoso (fibroma desmoplásico) é um tumor de tecido mole raro de origem fibroblástica. Na mucosa oral apenas dois casos foram descritos pela literatura. Nós descrevemos o caso de uma mulher de 42 anos de idade, cuja queixa era uma lesão indolor, de crescimento lento sob sua prótese total superior. Os achados histológicos da lesão incluíram fibroblastos escassos, estrelados ou fusiformes, dispersos em um estroma rico em colágeno. As células lesionais foram positivas para vimentina, mas rara marcação foi observada para actina músculo liso e fator XIIIa. A excisão cirúrgica total foi realizada e a recidiva da lesão não é esperada.

Objective: Mucoepidermoid carcinoma (MEC) is the most common malignant salivary tumour, classified as low, intermediate and high grade. Myofibroblasts are the main stromal component and are included as prognostic factor in some tumours. The aim of this study was to evaluate the myofibroblasts in the stroma of MEC with possible relationship to malignancy grading. Methods: Twenty-five cases of MEC (six low grade, 11 intermediate grade, four high grade and four metastasis) were stained for vimentin, desmin and smooth muscle actin (SMA) for the identification of myofibroblasts. Transforming growth factors (TGFβ1 and TGFβRII) were also assessed in our study. Results: Myofibroblasts were present in all cases, in amounts varying according to histological grading. TGFβ1 was positive in squamous cells of intermediate grade tumours, and in the stroma of only four cases. TGFβRII was positive in most squamous and intermediate cells, regardless of malignancy grading. Conclusions: Our study showed that the analysis of neoplastic stroma must be added to the studies of neoplastic cells to draw a better picture leading to tumour diagnosis and prognosis.

We describe a case of asymptomatic swelling of B-leukemic infiltrate involving right side of hard palate of a 62-year old white woman. The swelling was soft, well delimited and covered by a smooth, regular, and normal color mucosa. Microscopically it was composed by infiltration of the small lymphocyte with clumped chromatin, round nucleus and small nucleolus. The lymphoid cells were CD45, CD20 and CD79α-positive. A semi-nested PCR demonstrated the presence of heavy immunoglobulin gene rearrangement (IgH) with monoclonal pattern. The patient underwent involved field radiotherapy and systemic chemotherapy, but died due to progressive disease 2 years after the hard palate infiltration was detected.

Background: Murine double minute 2 (mdm2) is a cellular protooncogene, which, in conditions of overexpression or amplification, is capable of inactivating the functions of p53, leading to tumorigenesis. Immunoexpression of mdm2 in salivary gland tumours was previously found; however, it was necessary to find out if mdm2 was overexpressed. The aim of this study was to analyse the mRNA expression of mdm2 in salivary gland neoplasms and to correlate it to immunoexpression of p53 and p21 proteins. Methods: Specimens of different salivary gland neoplasms were obtained from surgical resections, and cell lineages derived from these tissues were established. RNA extraction was performed and mRNA expression was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Cellular expression of p53 and p21 proteins was investigated by immunofluorescence technique. Results: Increased expression of mdm2 was found in the majority of cell lines analysed. Conclusions: Comparing all results, we postulated that overexpression of mdm2 is related to the tumorigenesis and/or tumour progression of salivary gland neoplasms.

Background: Actinic cheilitis (AC) is a widely recognized precancerous lesion of the lip. Varying degrees of epithelial dysplasia may be present. However, no studies have correlated epithelial changes with cytokeratin expression that might reflect the disordered maturation that is probably occurring. Methods: Thirty-four cases diagnosed as AC were classified according to dysplasia degree, and submitted to immunohistochemical staining for the detection of cytokeratins (CKs) 7, 8, 13, 14, 16 and 19. Normal mucosa adjacent to the lesions was also evaluated. Results: The results obtained showed that CK10 immunostained only superficial keratinized epithelial layers in 11 cases, and also intermediate spinous layers in 18 cases. Cytokeratin 14 was expressed in all epithelial layers of 31 cases, in two cases its expression was in the basal and intermediate layers, and one case was negative. Cytokeratin 13 immunostained 26 cases and was negative in eight cases. In these eight cases, CK13 was apparently replaced by CK16. Cytokeratin 16, besides these eight cases, was also expressed in the spinous intermediate layers of a further eight cases. The remaining CKs tested were all negative. No relation between the degree of dysplasia and the CK expression was noted. Conclusions: Cytokeratin expression in AC is different from that of normal oral mucosa, and is not related to the degree of dysplasia.

Homeobox genes are regulatory genes encoding nuclear proteins that act as transcription factors, regulating aspects of morphogenesis and cell differentiation during normal embryonic development of several animals. Vertebrate homeobox genes can be divided in two subfamilies: clustered, or HOX genes, and nonclustered, or divergent, homeobox genes. During the last decades, several homeobox genes, clustered and nonclustered ones, were identified in normal tissue, in malignant cells, and in different diseases and metabolic alterations. Homeobox genes are involved in the normal teeth development and in familial teeth agenesis. Normal development and cancer have a great deal in common, as both processes involve shifts between cell proliferation and differentiation. The literature is accumulating evidences that homeobox genes play an important role in oncogenesis. Many cancers exhibit expression of or alteration in homeobox genes. Those include leukemias, colon, skin, prostate, breast and ovarian cancers, among others. This review is aimed at introducing readers to some of the homeobox family functions in normal tissues and especially in cancer.

Aims Metallothioneins (MTs) are proteins associated with the carcinogenesis and prognosis of various tumours. Previous studies have shown their potential as biomarkers in oral squamous cell carcinoma (OSCC). Aiming to understand more clearly the function of MTs in OSCC we evaluated, for the first time, the gene expression profile of MTs in this neoplasm. Materials and results Tissue samples from 35 cases of tongue and/or floor of mouth OSCC, paired with their corresponding non-neoplastic oral mucosa (NNOM), were retrieved (2007–09). All tissues were analysed for the following genes using TaqMan® reverse transcription–quantitative polymerase chain reaction (RT–qPCR) assays: MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT3 and MT4. The expression of MT1B and MT1H was seldom detected in both OSCC and NNOM. A significant loss of MT1A, MT1X, MT3 and MT4 expression and gain of MT1F expression was observed in OSCC, compared to NNOM. Cases with MT1G down-regulation exhibited the worst prognoses. The up-regulation of MT1X was restricted to non-metastatic cases, whereas up-regulation of MT3 was related to cases with lymph node metastasis. Conclusions Metallothionein mRNA expression is altered significantly in oral squamous cell carcinomas. The expression of MT1G, MT1X and MT3 may aid in the prognostic discrimination of OSCC cases.

The chicken inner ear is a remarkably complex structure consisting of eight morphologically distinct sensory organs. Unraveling how these sensory organs are specified during development is key to understanding how such a complex structure is generated. Previously, we have shown that each sensory organ in the chicken inner ear arises independently in the rudimentary otocyst based on Bone morphogenetic protein 4 (Bmp4) expression. Here, we compare the expression of Bmp4 with two other putative sensory organ markers, Lunatic Fringe (L-fng) and chicken Serrate1 (Ser1), both of which are components of the Notch signaling pathway. L-fng and Ser1 expression domains were asymmetrically distributed in the otic cup. At this early stage, expression of L-fng is similar to Delta1 (Dl1), in an anteroventral domain apparently corresponding to the neurogenic region, while Ser1 is expressed at both the anterior and posterior poles. By the otocyst stage, the expression of both L-fng and Ser1 largely coincided in the medial region. All presumptive sensory organs, as identified by Bmp4 expression, arose within the broad L-fng- and Ser1-positive domain, indicating the existence of a sensory-competent region in the rudimentary otocyst. In addition, there is a qualitative difference in the levels of expression between L-fng and Ser1 such that L-fng expression was stronger in the ventral anterior, whereas Ser1 was stronger in the dorsal posterior region of this broad domain. This early difference in expression may presage the differences among sensory organs as they arise from this sensory competent zone.

A mature inner ear is a complex labyrinth containing multiple sensory organs and nonsensory structures in a fixed configuration. Any perturbation in the structure of the labyrinth will undoubtedly lead to functional deficits. Therefore, it is important to understand molecularly how and when the position of each inner ear component is determined during development. To address this issue, each axis of the otocyst (embryonic day 2.5, E2.5, stage 16-17) was changed systematically at an age when axial information of the inner ear is predicted to be fixed based on gene expression patterns. Transplanted inner ears were analyzed at E4.5 for gene expression of BMP4 (bone morphogenetic protein), SOHo-1 (sensory organ homeobox-1), Otx1 (cognate of Drosophila orthodenticle gene), p75NGFR (nerve growth factor receptor) and Msx1 (muscle segment homeobox), or at E9 for their gross anatomy and sensory organ formation. Our results showed that axial specification in the chick inner ear occurs later than expected and patterning of sensory organs in the inner ear was first specified along the anterior/posterior (A/P) axis, followed by the dorsal/ventral (D/V) axis. Whereas the A/P axis of the sensory organs was fixed at the time of transplantation, the A/P axis for most non-sensory structures was not and was able to be re-specified according to the new axial information from the host. The D/V axis for the inner ear was not fixed at the time of transplantation. The asynchronous specification of the A/P and D/V axes of the chick inner ear suggests that sensory organ formation is a multi-step phenomenon, rather than a single inductive event.

Homeobox-containing genes encode a class of proteins that control patterning in developing systems, in some cases by acting as selector genes that define compartment identity. In an effort to demonstrate a similar role for such genes during ear development in the chicken, we present a detailed expression study of two related homeobox-containing genes, SOHo-1 and GH6, using in situ hybridization. At otocyst stages the two genes define a broad lateral domain of expression, which may represent a developmental compartment. Three-dimensional computer reconstructions of SOHo-1 expression at these and later stages revealed that the lateral domain becomes progressively restricted to the three semicircular canals. Thus, SOHo-1 and GH6 are among a small group of markers for a specific structural component of the inner ear. The gene expression domain initially includes the sensory regions of the semicircular canals, known as the cristae ampullaris, but none of the other four sensory organs which were recognizable by BMP4 expression during early morphogenesis (stages 19–24). Significantly, two of the sensory organs (the superior and posterior cristae) were found at the limits, or boundaries, of the SOHo-1/GH6 expression domain, suggesting that compartment boundaries may be involved in specifying sensory organ location as well as identity. Maintained expression at the boundaries may aid in specifying the location of canal outgrowth. These concepts are presented as a formal model which emphasizes that patterning information could be provided at the boundaries of gene expression domains in the inner ear.

Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.