Avaliação da incorporação de enxerto osseo homogeno fresco congelado em humanos: análise histologica e imuno-histoquímica

Allogeneic, fresh-frozen bone has been used in order to replace bone autografts. However, its osteoinduction and osteoconduction properties are not well-defined in the scientific literature. This work aimed to evaluate samples of homogenous bone grafts in humans by qualitative histological and immunohistochemical analysis. For this, ten pre-selected patients underwent surgical augmentation of bone defects. The homogenous fresh frozen block bone graft was stabilized and fixed by bicortical screws. After six months, the reopening procedure was performed for installation of osseointegrated implants. At this time surgical bone graft samples were removed by means of drill trephine. The samples were fixed in 10% formalin, processed with decalcified paraffin, and stained with hematoxylin and eosin. Immunohistochemistry was performed for the expression of Caspase 3 enzyme. The slides were brought to light microscopy for qualitative histology and immunohistochemistry. The results showed non-vital bone tissue, with few areas of deposition of new bone formation on the amorphous matrix, presence of chronic inflammatory infiltrate with areas of osteomyelitis, and expressive immunolabeling of Caspase 3. Given the methods employed and the results it was concluded that the allograft fresh-frozen block is not incorporated into the recipient bed after a healing period of six months.

Análise integrada entre dados de expressão global de transcritos codificadores e de miRNAs em carcinomas de células escamosas de laringe

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

MicroRNoma do carcinoma de pâncreas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of ATM and PTEN in prostatic carcinogenic dog prostate: validation of aCGH results

The canis lupus familiares is the only species besides human that spontaneously develop prostatic carcinoma (PCa). In addition, the metastatic sites are similar to those frequently reported in men. For these reasons, the dog is the best natural model to study the molecular mechanisms in PCa development providing a natural animal model for treatment by molecular targets. Previously, we investigated copy number alterations by arrayCGH (Canine Genome CGH Microarray 4x44K-G2519F, Agilent Technologies) in canine prostatic lesions: 3 benign prostatic hyperplasias (BPH), 4 proliferative inflammatory atrophies (PIA), and 14 PCa. Five histologically normal prostatic tissues were used as reference. Genomic alterations were evaluated using Genomic Workbench Standard Edition 5.0.14. This previous study revealed significant copy number losses of Atm and Pten exclusively in PCa. In the present study, ATM and PTEN immunoexpression were investigated using a tissue microarray (TMA) containing 149 canine prostatic paraffin-embedded lesions (BPH, PIA and PCa) collected from 67 animals. Immunohistochemical reactions were performed using the polyclonal rabbit antibody anti-PTEN (Santa Cruz Biotech, 1:50) and anti-ATM (Abcam, 1:50). The sections were developed with diaminobenzidine (DAB) and peroxidase. The immunohistochemical staining was assessed in each core by the distribution of positive cells for each antibody per lesion (score 1: <25% cells positive, 2: 26% to 50%, 3: being 51% and 75% and 4:> 75%) and intensity (1: weak, 2: moderate, 3: intense). Chi-square or Fisher exact test was used to determine the association between the categorical variables using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Distribution of positive cells did not differ among lesions. PCa and PIA showed more samples with weak intensity for ATM when compared to normal prostatic tissue and BPH (PCa: p=0,032 and PIA: p=0,025). Benign prostatic hyperplasia and normal samples presented intense PTEN immunostaining than PCa (p=0,021) and PIA (p=0,0013). These results suggest that ATM and PTEN proteins expression in canine prostatic carcinoma are downregulated possibly by copy number losses. These findings are similar from those described in prostate carcinomas from human corroborating for the use of dogs as a natural model to study prostatic disease in men.

Genomic gains in prostatic carcinoma and proliferative inflammatory atrophy in dogs

The dog can spontaneously develop prostate cancer and consequently can be used as an experimental model for prostatic diseases associated with aging, including benign prostate hyperplasia (BPH) and prostate carcinoma (PCa). DNA copy number variations (CNVs) have been used to identify genes associated with cancer development and progression. DNA microarray based comparative genomic hybridization (aCGH) is a technique that allows to identify copy number of thousands of genes throughout the genome. aCGH was used to identify genomic regions with significantly different DNA copy number in three benign prostatic hyperplasia (BPH), four proliferative inflammatory atrophy (PIA), and 14 canine prostate carcinoma (PCa). Five histologically normal prostate tissue were used as reference. Genomic DNA was extracted from formalin fixed and paraffin embedded samples and CNVs data was evaluated in Canine Genome CGH Microarray 4x44K (G2519F, Design ID021193, Agilent). Data analysis was performed using Genomic Workbench Standard Edition 5.0.14 (Agilent). PCa showed higher number of altered genes related to canonical diseases process, cellular functions and molecular pathways as well as greater inter-relationship between genes, compared with PIA and BPH. In conclusion, PCa showed a more complex genotype, being losses the most frequent genomic changes. Some discrepancies between genomic alterations in human and canine carcinomas may indicate the different clinical behavior of these tumors in these two species. In addition, it was observed was an ascending pattern of genomic complexity in BPH, PIA and CA consistent with a model of multistep tumor progression.

Métodos de determinação da densidade do solo em diferentes sistemas de manejo

Soil bulk density is an attribute often used to characterize soil physical structure, being an indicator of soil compaction. The objective of this study was to compare the values ​​of bulk density measured by the paraffin sealed clod and volumetric ring methods in conventional, no-tillage and minimum tillage systems on a Dystroferric Red Nitosol, clayey, in Botucatu, SP. The experiment design was a 3x2 factorial arrangement in randomized block with four replications. The density values ​​obtained by the paraffin sealed clod method were statistically higher than those obtained by the volumetric ring method. There was no difference between the managements when comparing the values ​​of soil density obtained by the paraffin sealed clod method. The soil under conventional management showed lower density when comparing the values ​​of soil density obtained by the volumetric ring. The volumetric ring method was more sensitive to show differences between the management systems than the paraffin sealed clod method.

Biocompatibilidade e bioatividade de cimentos à base de silicato tricálcico: estudo in vitro e in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resposta dos tecidos periodontais aos cimentos de silicato tricálcico associado ao óxido de zircônio ou óxido de nióbio usados no selamento de perfurações de furca em molares de ratos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gonadal Maturation in the Cobia Rachycentron canadum from the Northcentral Gulf of Mexico

Gonadal maturation of cobia, Rachycentron canadum, was evaluated by examining 508 specimens from its recreational fishery. Specimens were collected off southeast Louisiana to northwest Florida by hook-and-line during February through October 1987-1991. Fork lengths (FL) of these fish ranged from 580-1,530 mm, with corresponding weights of 2.0-43.5 kg. The female:male ratio was 1:0.37. Using a combination of oocyte size frequency and histological assessment of many of the fish, we determined that females were ripe from May through September, with atretic oocytes occurring in some fish from July through October. Degenerating hydrated oocytes in July and October and the presence of resting ovaries in July suggest two major spawning periods; however, monthly gonosomatic indices peaking in May, followed by a steady decline, do not support that finding. Ovaries were placed into undeveloped, early developing, mid-developing, or late developing categories based upon oocyte size-frequency distributions. Developing ovaries had two or three modes of oocytes larger than 30 μm. Batch fecundity was estimated to be 2.6×106 - 1.91×108 oocytes, depending on the size of fish/ovaries. The smallest female with oocytes exhibiting vitellogenesis was 834 mm FL. This fish was 2 years old based its otolith evaluation. The smallest male with an abundance of spermatozoa in its testes was 640 mm FL and 1 year old based on otolith evaluation; smaller males were not examined. Females larger than 840 mm FL had vitellogenic oocytes in March and April. A few fish still had vitellogenic oocytes in early October, but none did by late October. When Gilson’s fluid was used to assess ovarian tissue, the fresh weight of the tissue was reduced by 20% after being stored for 3 months. The diameter of oocytes shrunk about 25% in Gilson’s fluid which was 11% less than those fixed in formalin, embedded in paraffin, and sectioned. Tissue sections from specific individuals, each demonstrating a variety of different developmental stages, were similar regardless of whether they were obtained from the anterior, middle, or posterior portion of either ovary.

Biocompatibilidade e bioatividade de cimentos à base de silicato tricálcico: estudo in vitro e in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resposta dos tecidos periodontais aos cimentos de silicato tricálcico associado ao óxido de zircônio ou óxido de nióbio usados no selamento de perfurações de furca em molares de ratos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

FGFR4 Profile as a Prognostic Marker in Squamous Cell Carcinoma of the Mouth and Oropharynx

Background: Fibroblast growth factor receptor 4 (FGFR4) is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP) Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis. Patients and Methods: DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC) on paraffin-embedded tissue microarrays. Results: Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death. Conclusion: Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

TWIST and p-Akt immunoexpression in normal oral epithelium, oral dysplasia and in oral squamous cell carcinoma

Objectives: The aim of this study was to evaluate the immunoexpression of TWIST and p-Akt proteins in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), correlating their expressions with the histological features of the lesions. Study design: Immunohistochemical studies were carried out on 10 normal oral epithelium, 30 OL and 20 OSCC formalin-fixed, paraffin-embedded tissue samples. Immunoperoxidase reactions for TWIST and p-Akt proteins were applied on the specimens and the positivity of the reactions was calculated for 1000 epithelial cells. Results: Kruskal-Wallis and Dunn's post tests revealed a significant difference in TWIST and p-Akt immunoexpression among normal oral mucosa, OL and OSCC. In addition, a significant positive correlation was found between TWIST and p-Akt expressions according to the Pearson's correlation test. Conclusions: The results obtained in the current study suggest that TWIST and p-Akt may participate of the multi-step process of oral carcinogenesis since its early stages.

Topoisomerase expression in oral squamous cell carcinoma: relationship with cancer stem cells profiles and lymph node metastasis

BACKGROUND: The relationship between predictive proteins and tumors presenting cancer stem cells (CSCs) profiles in oral tumors is still poorly understood. This study aims to identify the relationship between topoisomerases I, II alpha, and III alpha and putative CSCs immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. METHODS: The following data were retrieved from 127 patients: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, and survival. An immunohistochemical study for topoisomerases I, II alpha, and III alpha was performed in a tissue microarray containing 127 paraffin blocks of OSCCs. RESULTS: In univariate analysis, topoisomerases expression showed significant differences according to CSCs profiles and p53 immunoexpression, but not with survival. Topoisomerases II alpha and III alpha also showed significant relationship with lymph node metastasis. The multivariate test confirmed these associations. CONCLUSIONS: The results that all topoisomerases correlates with OSCC CSCs may indicate a role for topoisomerases in head and neck carcinogenesis. Notwithstanding, it is plausible that other members of topoisomerases family could represent novel therapeutical targets in oral squamous cell carcinoma. J Oral Pathol Med (2012) 41: 762-768