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The relationship between the histological grading of malignancy and the expression of vimentin and cytokeratin was studied in 43 cases of oral squamous cell carcinoma. Immunohistochemical analysis was carried out with the avidin-biotin method using monoclonal antibody anti-vimentin, and the peroxidase anti-peroxidase method using the polyclonal antibody anti-cytokeratin. All cases were classified according to the histological malignancy grading system proposed by Anneroth. All of the carcinomas were found to express cytokeratin, while 60.4% expressed vimentin. Vimentin was particularly noted in all tumors scored to have highly malignant cells.

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

Quando se encontram remanescentes humanos, algumas questões sobre a identificação são levantadas e uma das primeiras diz respeito à antiguidade do material. A distinção entre amostras recentes e arqueológicas pode ser feita pela identificação ou não de fungos na dentina humana através da técnica histoquímica do PAS (periodic acid Schiff). Para validar esta técnica, avaliamos três amostras diferentes. A primeira composta de dentes de um indivíduo com datação de 2640 anos antes da data presente. A segunda composta de dentes com idades de 69, 67, 48 e 36 anos. A terceira refere-se a dois dentes extraídos recentemente. Na amostra mais antiga observou-se a presença de inúmeras estruturas filamentosas compatíveis com hifas de fungos no interior dos túbulos dentinários. Estes resultados apontam a presença dessas estruturas na dentina como um indicativo de que o material não tem origem recente. A técnica utilizada é um recurso de baixo custo e rápidos resultados para uma primeira classificação e distinção dos remanescentes humanos.

Objective TGIF1 homeobox gene involvement in oral cancer has not yet been investigated. This study analyzed the expression of TGIF1 transcripts and protein in oral squamous cell carcinoma (OSCC). Study design Snap-frozen samples from 16 patients were taken from both OSCC and nontumoral adjacent epithelium (NT) for in situ hybridization (ISH). Forty-six paraffin-embedded samples of OSCC were submitted to immunohistochemistry (IHC). A descriptive analysis of the transcript signal detection was accomplished, and TGIF1 immunoexpression was carried out considering protein levels, localization, and cellular differentiation. Results ISH reactions showed TGIF1 transcripts with a signal that was frequently intense in NT, and generally weak in OSCC, and that had stronger transcript signal in well-differentiated areas of OSCC when compared with poorly differentiated ones. IHC reactions had poorly differentiated cases associated with TGIF1 protein expression in both the nucleus and cytoplasm (P = .05, Fisher test). Conclusions TGIF1 gain or loss of function might possibly play a role in oral cancer cell differentiation.

Patients who undergo hematopoietic stem cell transplantation (HSCT) frequently experience gastrointestinal toxicity as a result of their preparative regimen. The most frequent manifestation is oral mucositis (OM) and diarrhea. We studied the effects of oral care prior to HSCT on the severity of OM. Seventy patients suffering from hematologic malignancies who had undergone HSCT were divided into two groups (35 patients - Study Group [SG] and 35 - Control Group [CG]), and the severity of OM was evaluated by two calibrated dentists, using the WHO scale. The patients from the SG received oral care prior to HSCT and those from CG did not receive any dental care. The results showed no differences (p = 0.20) in the incidence or severity of OM among the groups. However, patients from the SG presented a shorter time elapsed (p0.001) when compared with the CG (median: 10 vs. 20 d). Our results show the importance of simple, inexpensive preventive intervention to control the time elapsed of OM, which reduces morbidity and, as a consequence, the cost of the treatment.

Glandular Odontogenic Cyst (GOC) is a rare developmental cyst of the jaws. The histological features of GOC strongly suggest an origin from the remains of dental lamina. Radiographically, GOC presents as well-defined radiolucencies with uni- or multilocular appearance. A case of GOC in a 54-year- old black female is presented here. Clinical, histological and imaging features were evaluated. Due to the high tendency of recurrence and the aggressive potential of GOC, careful clinical and radiological evaluation must be carried out. CT scans are recommended because they provide accurate information about locularity of the lesion, cortical integrity, expansion of the lesion and involvement of the contiguous soft tissue. (J Oral Sci 51, 467-470, 2009)

Made available in DSpace on 2019-09-12T16:53:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) This study examined the clinical, histological, and immunohistochemical features as well as the differential diagnoses of oral intravascular papillary endothelial hyperplasia (IPEH) to aid clinicians and pathologists in its diagnosis. Clinical features of five oral IPEH cases were obtained from medical records, and all histopathological diagnoses were reviewed. Immunohistochemical reactions, including anti-CD-34, laminin, vimentin, estrogen receptor alpha, and Ki-67, were assessed. Microscopically, a reactive proliferation of vascular cells composed of small papillary structures with hypocellular and hyalinized cores arising in an organized thrombus was seen. CD-34, vimentin, and laminin staining were strongly positive, while estrogen receptor alpha was negative in all cases. A low percentage of cells were positive for Ki-67 in four of five lesions, but one case was strongly positive. A diagnosis of angiosarcoma was investigated and rejected. IPEH presents specific microscopic characteristics that along with clinical data lead to an accurate diagnosis. The general dentist, the first to participate in the diagnostic process, must share the responsibility for diagnosis with the pathologist, and they must work together to determine the correct diagnosis and management. Oral lesions of IPEH are uncommon. Their main significance is that they show a microscopic resemblance to angiosarcoma. Thus, clinicians should have more information regarding this benign entity. Finally, we suggest that in recurrent cases exhibiting strong immunolabeling of proliferative markers the possibility of angiosarcoma should be investigated. [Campos, Marcia Sampaio] Universidade de Taubaté (Unitau), Dent Grad Program, Dept Dent, BR-12020330 Taubate, SP, Brazil [Fontes, Alexandra; Campos, Marcia Sampaio; de Sousa, Suzana C. O. M.; Martins, Marilia Trierveiler; Nunes, Fabio Daumas] Univ Sao Paulo, Dept Oral Pathol, Sch Dent, Sao Paulo, Brazil

AIM: To investigate the immunoexpression and diagnostic applicability of human erythrocyte-type glucose transporter protein (GLUT-1) in oral peripheral nerve sheath tumors. MATERIAL AND METHODS: Specimens diagnosed as oral peripheral nerve sheath tumors archived in the Oral Pathology Service of Universidade Federal de Minas Gerais from 1966 to 2006 were evaluated. Thirty-four lesions were included: 15 traumatic neuromas, 11 neurofibromas, four neurilemmomas, and four malignant peripheral nerve sheath tumors (MPNST). One case of neurofibroma was associated with neurofibromatosis type I. Immunohistochemistry for S-100 and GLUT-1 was performed. S-100 was immunopositive in all lesions. RESULTS: Benign lesions were immunopositive for GLUT-1 except in two (18.2%) cases of neurofibromas. In the traumatic neuroma, the perineuriums were immunopositive for GLUT-1. In the neurofibroma, the immunoreactivity was heterogeneous. Immunopositivity was observed at levels of 54.5% in the periphery of the lesion, 9.1% in the center, and 18.2% in both. The neurilemmoma demonstrated immunopositivity in the capsule. One case (25%) of MPNST presented GLUT-1 positive stain in occasional cells distributed homogeneously in all the tumor area. CONCLUSION: GLUT-1 is a useful marker for perineurial cells and should be included in the oral peripheral nerve sheath tumors immunophenotyping thus aiding in the correct diagnosis of these lesions. Oral Diseases (2008) 14, 510–513

The selection of housekeeping genes is critical for gene expression studies. To address this issue, four candidate housekeeping genes, including several commonly used ones, were investigated in oral squamous cell carcinoma cell lines. A simple quantitative RT-PCR approach was employed by comparing relative expression of the four candidate genes within two cancerous cell lines (HN6 and HN31) and one noncancerous cell line (HaCaT) treated or not with EGF and TGF-beta1. Data were analyzed using ANOVA followed by the NormFinder software program. On this basis, stability of the candidate housekeeping genes was ranked and non statistical differences were found using ANOVA test. On the other hand, the NormFinder was able to show that GAPD and TUBB presented the less variable results, representing appropriated housekeeping genes for the samples and conditions analyzed. In conclusion, this study suggests that the GAPD and the TUBB represent adequate normalizers for gene profiling studies in OSCC cell lines, covering, respectively, high and low expression levels genes.

Generalized aggressive periodontitis (GAP) comprises a group of periodontal diseases characterized by the rapid destruction of periodontal tissues which affect young individuals who generally present no systemic disorders. Polymorphisms in the interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) genes have been associated with an increased severity of chronic periodontitis. The objective of the present study was to evaluate the association between IL-1A (-889) and TNFA (-308) gene polymorphisms and GAP. One hundred nonsmoking subjects were selected, including 30 with GAP and 70 without periodontal disease. Gene polymorphisms were analyzed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. For IL-1 (-889), the frequency of genotype 1/1 was 54.3% in the control group and 56.7% in the study group. The frequency of genotype 1/2 was 37.1% in the control group and 40% in the study group. Genotype 2/2 was detected at a frequency of 8.6% and 3.3% in the control and study groups, respectively. For TNFA, genotype 1/1 was present in 68.6% of control subjects and in 80.0% of patients with GAP, while the frequency of genotype 1/2 was 27.1% in the control group and 20% in the study group. Genotype 2/2 was present in 4.3% of control subjects and was not detected in the study group. The frequencies of allele 1 and allele 2 of the IL-1A (-889) gene were 72.9% and 27.1%, respectively, in the control group and 76.7% and 23.3% in the GAP group. For the TNFA (-308) gene, the frequency of allele 1 was 82.15% in the control group and 90% in the study group, whereas the frequency of allele 2 was 17.85% in the control group and 10% in the study group. Statistical analysis revealed no significant difference in allele distribution for either gene between the two groups. No association was observed between GAP and IL-1A (-889) and TNFA (-308) gene polymorphisms in Brazilian patients.

A comparative nonisotopic in situ hybridization (ISH) analysis was carried out for the detection of Bmp-4, Shh and Wnt-5a transcripts during mice odontogenesis from initiation to cap stage. Bmp-4 was expressed early in the epithelium and then in the underlying mesenchyme. Shh expression was seen in the odontogenic epithelial lining thickening, being stronger in the enamel knot area, during the cap stage. Wnt-5a transcripts were expressed only in the mesenchyme during the initiation, bud and cap stages, with strong expression in the dental mesenchyme during the bud stage. The present results showed that Bmp-4, Shh and Wnt-5a are expressed since the very early stages of tooth development, and they suggest that the Wnt-5a gene is expressed in different cell populations than Bmp-4 and Shh.

metastasis. The purpose of this study is to clarify the direct role of VEGF and its receptor Flt-1 signaling in oral squamous cell carcinoma (OSCC) progression. Study Design: Proliferation, Migration and Invasion abilities of OSCC cells and their signaling pathways were examined in high and non-Flt-1 expressing cells (HSC2 and HO1-N1, respectively) by Flt-1 specific ligand-placental growth factor (PlGF) stimulation. Results: Low concentration of PlGF stimulation induced slight up-regulation of the proliferation and promoted the migration and invasion of HSC2, while it showed no effect on HO1-N1. Interestingly, PlGF also up-regulated MMP-1 and MMP-9 expression via phosphorylation of p38 and ERK1/2 in HSC2. Conclusions: VEGF-Flt-1 signaling plays an important role in OSCC progression by facilitating invasion and migration of OSCC through MMP-1 and MMP-9 production and blocking of VEGF-Flt-1 signaling may be beneficial for the treatment of OSCC.

The aim of this study was to investigate loss of heterozygosity (LOH) of the APC tumor suppressor gene loci, using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in 40 cases of oral squamous cell carcinoma (OSCC). Observed informativity was 72.5% for APC exon 11 and 82.5% for APC exon 15. LOH at APC exon 11 was observed in 2 (6.9%) of 29 informative cases, and no LOH was observed for APC exon 15. Our results suggest that inactivation of the APC gene plays a minor role in the carcinogenesis of OSCC.

Background: Homeobox are a family of developmental genes involved in morphogenesis and cellular differentiation. Participation of homeobox within normal and malignant tissue has been recently discussed in the literature. Objective: To analyze the presence of HOXB13 transcript expression in human minor salivary gland. Material and methods: Ten-micrometer sections from frozen samples were evaluated employing non-radioactive in situ hybridization technique and HOXB13 mRNA probes. Results: HOXB13 was found to be expressed in ducts and mucous acini but not in serous acini. Conclusions: Results suggest that HOXB13 transcripts are differently expressed in normal mucous and serous acini, and it may possibly reflect a different role in salivary gland carcinogenesis.

A técnica de hibridização in situ (ISH) tem sido usada para identificar mRNA (ou DNA) em amostras de tecido de material humano e animal. Embora uma série de protocolos para essa técnica seja utilizada, as descrições não são bem detalhadas. O objetivo deste trabalho é descrever a reação de hibridização in situ em tecido fresco e sua aplicação em patologia, tornando mais compreensível essa técnica tão importante, que possibilita observar a localização tecidual e a expressão temporal e espacial dos transcritos de um determinado gene (mRNA). Resultados de reações com as ribossondas PITX1, SHH e WNT-5A, realizadas em amostras de tecido congelado, são apresentados.

Human Papilomaviruses (HPV) are a group of viruses associated with benign and malignant lesions of cutaneous and mucosal epithelia. Some "high risk" HPV types, especially HPV 16 and 18, are strongly correlated with cervical and anogenital cancers and are also related to the genesis of oral squamous cell carcinomas (OSCC). The aim of this work was to investigate the incidence of HPV infection in 40 paraffin-embedded or fresh specimens of OSCC, using PCR amplification of the viral DNA. Literature based primers (GP5+/GP6+) were used in order to amplify HPV DNA from the L1 gene, present in more than 22 types of HPV. A condyloma case with HPV 16 and 18 detected by in situ hybridization was used as a positive control. Amplification of HPV was observed only in the positive control. No squamous cell carcinoma cases showed DNA viral amplification. Absence of HPV DNA amplification by PCR in the analyzed specimens of OSCCs suggests that this virus not always plays a role in the carcinogenesis process. Discrepancy with some studies found in the literature may be related to methodology or population differences.

Oral hairy leukoplakia (OHL) is a lesion associated with a compromised immune system, and its diagnosis is determined by the demonstration of the presence of Epstein-Barr virus (EBV) in lesional tissue. The purpose of this article was to develop a simple technique to help the diagnosis of OHL, using PCR as an alternative technique to evidence EBV in scrapings. DNA samples were obtained by scraping the lateral border of the tongue of 38 adult patients: 29 HIV-positive patients (4 with clinical evidence of OHL; 4 with history of OHL, but without lesion at the moment the samples were collected; and 21 without clinical evidence of OHL), and 9 healthy volunteers for the control group. DNA was extracted from scrapes and amplified by PCR using specific primers for EBV. Of the 29 cases of HIV-positive patients, 22 (75.86%) were positive for EBV: 2 patients with clinical evidence of OHL, 4 patients with history of OHL, but without lesion at the moment the samples were collected, and 16 patients without clinical evidence of OHL. In the control group, samples of 5 (55.56%) healthy volunteers presented amplification for EBV. We concluded that the use of PCR in oral scrapes suggests a high sensitivity but low specificity for the diagnosis of OHL. A leucoplasia pilosa (LP) é uma lesão associada ao comprometimento do sistema imune e seu diagnóstico é determinado pela demonstração da presença do vírus Epstein-Barr (EBV) no tecido lesionado. O objetivo deste trabalho foi desenvolver uma metodologia simples para auxiliar no diagnóstico da LP, utilizando-se a técnica da PCR como uma alternativa para evidenciar o EBV em esfregaços da lesão. Amostras de DNA foram obtidas por meio de raspado de borda lateral de língua, de 38 pacientes adultos, sendo estes: 29 pacientes HIV positivos (4 com evidência clínica de leucoplasia pilosa, 4 que haviam apresentado LP previamente, mas não no momento da coleta, e 21 sem evidência clínica de leucoplasia pilosa) e 9 voluntários sadios para o grupo controle. O DNA foi extraído do material obtido por raspagem e amplificado pela PCR utilizando-se iniciadores específicos para o EBV. Dos 29 casos de pacientes portadores do vírus HIV, 22 (75,86%) foram positivos para o EBV, sendo: 2 de pacientes com evidência clínica de LP, 4 de pacientes que haviam apresentado LP previamente e 16 de pacientes sem sinais clínicos de LP. No grupo controle, as amostras de 5 (55,56%) indivíduos clinicamente sadios mostraram amplificação para o EBV. Concluímos que o uso da PCR em esfregaços sugere uma alta sensibilidade e baixa especificidade no diagnóstico da leucoplasia pilosa.

Objective The aim of this study was to detect the chromosomal translocation t(12;16)(q13;p11) that leads to a gene fusion encoding a FUS-CHOP chimeric protein and has been shown to be highly characteristic of myxoid and round cell subtypes of liposarcoma, in a case of oral myxoid liposarcoma. Method and Materials Nested reverse transcriptase–polymerase chain reaction to detect the TLS/FUS-CHOP fusion gene transcript was performed. A case of inflammatory fibrous hyperplasia and a case of oral lipoma were included as negative controls. Results Only the myxoid oral liposarcoma showed a 103–base pair product, specific of TLS/FUS-CHOP fusion type II transcript. Conclusion The identification of FUS-CHOP transcript is potentially useful in the diagnosis and research of oral liposarcomas.

The purpose of this study was to determine the prevalence of Epstein-Barr virus (EBV)-DNA in the oral mucosa of renal transplant patients and observe the efficacy of mouth rinses with phosphate-buffered saline (PBS) to eliminate EBV present in the saliva. Lingual, gingival, and buccal cytobrushings were obtained from normal oral mucosa of 10 renal transplant patients and 10 normal subjects, and were examined through polymerase chain reaction (PCR), before and after rinses with PBS. EBV-DNA was detected in 86.6% of renal transplant recipients and in 46.6% of healthy subjects. No significant difference was observed between oral scrapes obtained before and after rinses with PBS with regard to detection of EBV-DNA. Our results suggest that the use of PCR to detect the presence of EBV-DNA in oral mucosa in the absence of specific lesions gives rise to the problem of identifying the viral replication sites. In addition, PBS was not effective at minimizing contamination by saliva. Diagn. Cytopathol. 2006;34:24–28. © 2005 Wiley-Liss, Inc.

INTRODUCAO: O material fixado em formol e embebido em parafina constitui hoje a maior fonte de tecido humano arquivado. O DNA extraido de tecido parafinado por vezes nao e adequado para as tecnicas de biologia molecular, visto que se apresenta parcialmente degradado. OBJETIVOS: O objetivo desse estudo foi verificar se tecido humano de boca parafinado e arquivado pelos ultimos 40 anos pode ser usado para a reacao em cadeia da polimerase (PCR). METODOS: Amostras foram submetidas a tecnica de extracao de DNA pelo metodo do fenol-cloroformio. Para quantificacao e qualificacao do DNA foram realizadas analise em espectrofotometro, eletroforese em gel de agarose e amplificacao pela tecnica da PCR. RESULTADOS: Foi observada fraca correlacao positiva entre a quantidade de DNA obtida e a idade das amostras. A eletroforese em gel de agarose demonstrou que a maioria do DNA obtido foi constituida de fragmentos pequenos. O fragmento de 268-pb do gene da b-globina foi amplificado em 55% dos casos, preferencialmente nos casos mais recentes. O fragmento de 149-pb do gene WAF1 apresentou amplificacao fraca, mas presente em 75% dos casos. O fragmento de 536-bp do gene da b-globina foi detectado em somente 25% dos casos e tambem preferencialmente nos casos mais recentes. CONCLUSOES: Esse estudo mostrou que, apesar de o DNA estar degradado, e possivel usar DNA genomico extraido de tecido parafinado arquivado pelos ultimos 40 anos em reacoes de PCR de produtos pequenos. A amplificacao de produtos maiores, entretanto, e mais dificil.

Many studies have demonstrated that loss of TP53 gene function has an important role in the genesis of many neoplasms, including salivary gland neoplasms. The purpose of this study was to examine the mutation profile of the TP53 gene in salivary gland neoplasms. Genomic DNA was extracted from paraffin-embedded tissues of pleomorphic adenoma, carcinoma in pleomorphic adenoma, mucoepidermoid carcinoma, adenoid cystic carcinoma and polymorphous low grade adenocarcinoma. Exons 5 to 8 of the TP53 gene were amplified by polymerase chain reaction (PCR) to perform single-stranded conformational polymorphism (SSCP) analysis. Band shifting was observed in exons 5, 6 and 8 in 9 out of 18 neoplasms. The results of this study suggest that mutations in TP53 gene are related to salivary gland neoplasms pathogenesis and that exons 5 and 8 are most frequently involved. Vários estudos mostram que a perda da função do gene TP53 desempenha um importante papel na gênese de diversas neoplasias, incluindo as neoplasias de glândula salivar. Assim, o objetivo deste estudo foi avaliar a presença de mutações no gene TP53 em neoplasias de glândula salivar. Para isso, DNA genômico foi extraído de casos de adenoma pleomórfico (AP), carcinoma em adenoma pleomórfico (CAP), carcinoma mucoepidermóide (CME), carcinoma adenóide cístico (CAC) e adenocarcinoma polimorfo de baixo grau de malignidade (APBG) emblocados em parafina. Foi realizada amplificação pela técnica da PCR dos exons 5 a 8 e em seguida a SSCP (análise de conformação de fita simples). Foi observada alteração na mobilidade das bandas em 9 das 18 neoplasias estudadas, principalmente nos exons 5 e 8. Esses achados sugerem que mutações no gene TP53 estão relacionadas à patogênese das neoplasias de glândula salivar e que os exons 5 e 8 estão mais freqüentemente envolvidos.

We compared the immunoexpression of 5 myoepithelial cell (MEC) markers (alpha-smooth-muscle actin, calponin, h-caldesmon, vimentin, and S-100-protein) using 16 pleomorphic adenomas (PA), 15 adenoid cystic carcinomas (ACC), and 3 epithelial-myoepithelial carcinomas (EMC) of salivary glands. The alpha-smooth-muscle actin was useful for identification of MECs, especially in cribriform and tubular ACC, followed by EMC. Calponin was similar to alpha-smooth-muscle actin, except for polygonal and plasmacytoid cells of PA and for solid ACC, which showed alpha-smooth-muscle actin negative and calponin positive. H-caldesmon was negative. Vimentin immunostained all MEC types, and was negative in luminal cells. S-100 protein was expressed both in the nuclei and cytoplasm of MECs and luminal cells, especially in PA. The best way to identify MEC is using alpha-smooth-muscle actin or calponin, plus vimentin, since in tumors MECs are hardly ever fully differentiated.

The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268bp fragments of APC and beta-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536bp fragment of beta-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR.

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.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 (TGFbeta1 and TGFbetaRII) were also assessed in our study.Myofibroblasts were present in all cases, in amounts varying according to histological grading. TGFbeta1 was positive in squamous cells of intermediate grade tumours, and in the stroma of only four cases. TGFbetaRII was positive in most squamous and intermediate cells, regardless of malignancy grading.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.

The expression of G1-phase cell-cycle regulators is commonly deregulated in human malignancies. In the present study, we investigate components of the retinoblastoma (RB) pathway in normal salivary glands (NSG) and in salivary gland tumours (SGT). Samples of NSG, pleomorphic adenoma (PA), adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), epithelial-myoepithelial carcinoma (EMC), malignant myoepithelioma (MEM), carcinoma ex pleomorphic adenoma (CEPA), and polymorphous, low-grade adenocarcinoma (PLGA) were examined immunohistochemically using antibodies to cyclin D1, cyclin-dependent kinase 4 (CDK-4), retinoblastoma protein (pRb), CDK inhibitor p16 and transcription factor E2F-1. In normal salivary glands, cyclin D1 and cdk-4 were not expressed in any case while p16 was positively expressed. pRb was abundant and E2F-1 moderately expressed. In tumors, cdk-4 was overexpressed in half of the cases. Most tumour cases showed decreased pRb immunoexpression compared to normal salivary glands. In contrast, expression of p16 and E2F-1 increased. pRb expression was absent in three cases of PA, two of EMC and one of CEPA. One case of MEM and one of PLGA showed no E2F-1 expression. Statistical analyses revealed positive correlations between cyclin D1 and cdk-4, cyclin D1 and E2F-1, cdk-4 and E2F-1, and p16 and E2F-1. The benign and malignant tumours expressed retinoblastoma pathway proteins differently form the normal salivary gland. Our findings suggest that, pRb pathway deregulation in salivary gland neoplasms is unrelated to their biological behaviour.

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples. Existem na literatura vários protocolos para extração de DNA genômico a partir de material fixado em formol e embebido em parafina. A obtenção de DNA genômico é importante para realização de experimentos em biologia molecular, dentre eles a PCR. Este trabalho teve por objetivo otimizar a extração de DNA genômico a partir de material fixado (hiperplasia fibrosa inflamatória) e não-fixado (mucosa bucal normal) em formol, comparando-se três metodologias diferentes: fenol com digestão enzimática, partículas de sílica com e sem digestão enzimática. Para amplificação do DNA pela técnica da PCR, utilizou-se iniciadores para o éxon 7 da citoqueratina humana tipo 14. O sucesso da amplificação foi verificado pela eletroforese do produto em gel de poliacrilamida 8% contendo glicerol 5% corado com prata. Obteve-se amplificação do DNA genômico extraído com fenol/digestão enzimática e com partículas de sílica/digestão enzimática, para ambos tecidos utilizados. O método padronizado tem potencial para auxiliar no diagnóstico histopatológico, assim como no estudo retrospectivo de material de arquivo.

Background Functional in vitro studies are essential for the understanding of the role of microRNAs, small noncoding RNA molecules that function as posttranscriptional regulators, in cancer. In this study we investigated the effect of the over-expression of microRNAs previously identified as deregulated in squamous cell carcinomas of the head and neck (miRNA-1, miRNA-7, miRNA-10b e miRNA-196a) in cell proliferation.

Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

We present the case of a 65-year-old woman who had a painless mass in the left buccal mucosa. Histology showed a benign osteolipoma.

on final consolidation are achieved with daily administration of 40 μg/kg of PTH1-34. Our results suggest the existence of a natural window of treatment efficacy in mouse peaking 18 d. after fracture. Healing processes are anticipated and enhanced from early teriparatide administration. Further applications of the study could be the treatment of osteoporotic or secondary fractures, consolidation delays and non-unions. Disclosure of Interest: None declared

Bone morphogenetic protein 4 (Bmp4) is expressed during multiple stages of development of the chicken inner ear. At the otocyst stage, Bmp4 is expressed in each presumptive sensory organ, as well as in the mesenchymal cells surrounding the region of the otocyst that is destined to form the semicircular canals. After the formation of the gross anatomy of the inner ear, Bmp4 expression persists in some sensory organs and restricted domains of the semicircular canals. To address the role of this gene in inner ear development, we blocked BMP4 function(s) by delivering one of its antagonists, Noggin, to the developing inner ear in ovo. Exogenous Noggin was delivered to the developing otocyst by using a replication-competent avian retrovirus encoding the Noggin cDNA (RCAS-N) or implanting beads coated with Noggin protein. Noggin treatment resulted in a variety of phenotypes involving both sensory and nonsensory components of the inner ear. Among the nonsensory structures, the semicircular canals were the most sensitive and the endolymphatic duct and sac most resistant to exogenous Noggin. Noggin affected the proliferation of the primordial canal outpouch, as well as the continual outgrowth of the canal after its formation. In addition, Noggin affected the structural patterning of the cristae, possibly via a decrease of Msx1 and p75NGFR expression. These results suggest that BMP4 and possibly other BMPs are required for multiple phases of inner ear development.