Canine Subcutaneous Mast Cell Tumors: Cellular Proliferation and KIT Expression as Prognostic Indices

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers-including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)-as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.

Concentração de flúor em águas engarrafadas comercializadas no município de São Paulo

O objetivo do estudo foi avaliar a concentração de flúor na água engarrafada comercializada, comparando-a aos valores impressos no rótulo da embalagem. Foram coletadas 229 amostras de água das 35 marcas disponíveis em diversos supermercados, mercearias e lanchonetes de grande circulação nas diferentes regiões do município de São Paulo, em 2006. A concentração de flúor foi determinada por análise em duplicata, utilizando o eletrodo íon-específico. A concentração de flúor variou entre 0,01 e 2,04 mg/l, com diferenças significantes entre os valores estipulados nos rótulos e os encontrados com a análise. Esses resultados reforçam a importância do controle dos níveis de flúor na água engarrafada por parte da vigilância sanitária.

Decrease in the number and apoptosis of alveolar bone osteoclasts in estrogen-treated rats

Bone is a mineralized tissue that is under the influence of several systemic, local and environmental factors. Among systemic factors, estrogen is a hormone well known for its inhibitory function on bone resorption. As alveolar bone of young rats undergoes continuous and intense remodeling to accommodate the growing and erupting tooth, it is a suitable in vivo model for using to study the possible action of estrogen on bone. Thus, in an attempt to investigate the possibility that estrogen may induce the death of osteoclasts, we examined the alveolar bone of estrogen-treated rats.Fifteen, 22-d-old female rats were divided into estrogen, sham and control groups. The estrogen group received estrogen and the sham group received corn oil used as the dilution vehicle. After 8 d, fragments containing alveolar bone were removed and processed for light microscopy and transmission electron microscopy. Sections were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP)-an osteoclast marker. Quantitative analysis of the number of TRAP-positive osteoclasts per mm of bone surface was carried out. For detecting apoptosis, sections were analyzed by the Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) method; TUNEL/TRAP combined methods were also used.The number of TRAP-positive osteoclasts per mm of bone surface was significantly reduced in the estrogen group compared with the sham and control groups. TRAP-positive osteoclasts exhibiting TUNEL-positive nuclei were observed only in the estrogen group. In addition, in the estrogen group the ultrastructural images revealed shrunken osteoclasts exhibiting nuclei with conspicuous and tortuous masses of condensed chromatin, typical of apoptosis.Our results reinforce the idea that estrogen inhibits bone resorption by promoting a reduction in the number of osteoclasts, thus indicating that this reduction may be, at least in part, a consequence of osteoclast apoptosis.

Cell death and quantitative reduction of rests of Malassez according to age

Background and Objective: Rests of Malassez are clusters of epithelial cells that remain in the periodontal ligament throughout life. However, it has been reported that the number of these structures decreases with age, and some epithelial cells undergo apoptosis in rests of Malassez of young and adult rats. Therefore, the purpose of the present study was to investigate the incidence of epithelial cell death and the quantitative changes in the rests of Malassez in rat molars of different ages.Material and Methods: Fragments containing the upper molars of rats aged 29, 45 and 120 d were fixed, decalcified and embedded for analysis by light microscopy. In the sections stained by hematoxylin and eosin, the number of rests of Malassez and the number of nuclei of these epithelial structures were obtained. Moreover, the nuclei exhibiting typical features of cell death were also counted in each rest of Malassez. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method for detection of cell death was also carried out.Results: In all groups examined, some rests of Malassez exhibited epithelial cell nuclei with typical features of apoptosis and some of them were also TUNEL positive. From 29 to 120 d of age in rats, the quantitative analysis showed a significant decrease in the total number of rests of Malassez in the cervical, middle and furcation regions of the periodontal ligament. Moreover, a significant decrease of epithelial cell nuclei was concomitant to an increase in the frequency of cell death in the oldest rats.Conclusion: These results suggest that epithelial cell death by apoptosis may be, at least in part, responsible for the reduction in the number of rests of Malassez according to age.

Vitamin B-12 Supplement Exerts a Beneficial Effect on the Seminiferous Epithelium of Cimetidine-Treated Rats

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

Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aquaporin 9 (AQP9) localization in the adult dog testis excurrent ducts by immunohistochemistry

Aquaporins (AQPs) are small, intrinsic membrane proteins that are present in many cell types involved in fluid transport. AQP9 is a major apical water channel that is expressed throughout the efferent ducts, epididymis, and vas deferens, as well as in other regions of the human and rodent male reproductive tract. The target of this study was to examine the expression of AQP9 in epithelial cells in the adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis; initial segment, caput, corpus, and cauda of the epididymis; and vas deferens were obtained from eight adult mongrel dogs. Immunohistochemistry and Western blotting procedures were used to show AQP9 localization and distribution. AQP9 expression was not detected either in dog seminiferous tubules or rete testis. However, apical labeling for AQP9 was detected in the different regions of epididymis and vas deferens, with the reaction being less intense in the caput epididymis. Thus, AQP9 is abundantly expressed in dog male reproductive tract, in which it is an important apical pathway for transmembrane flow of water and neutral solutes.

Localization of 5-methylcytosine in metaphase chromosomes of diploid and triploid pacu fish, Piaractus mesopotamicus (Pisces, Characiformes)

The distribution of 5-methylcytosine (5-MeC) was investigated in fish chromosomes by indirect immunofluorescence using a highly specific 5-MeC monoclonal antibody. Diploid and artificially produced triploid specimens of the pacu fish, Piaractus mesopotamicus, were analyzed. The strong immunofluorescent signals were coincident with the heterochromatic regions of both diploids and triploids in a pattern that matched the C-banding pattern. In the euchromatin, heterogeneous labeling was observed along the chromatids. The weakness of this labeling hindered comparison of the fluorescence labeling of homologous chromosomes from diploid and triploid individuals. However, no striking differences were observed. The possibility that the euchromatin labeling by the 5-MeC antibody is related to the occurrence of mildly repetitive sequences in the genome of Piaractus is discussed.

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.

Phosphorylation of Histone H3S10 in Animal Chromosomes: Is There a Uniform Pattern?

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133(+) stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10 (13) mol iron (9.5 pg) or 7.0 x 10 (6) nanoparticles per cell ( the measurement was carried out in a volume of 2 mu L containing about 6.16 x 10 5 pg iron, equivalent to 4.5 x 10 (11) SPIONs). (c) 2008 Elsevier B.V. All rights reserved.

The Ultrastructural Study of Tumorigenic Cells Using Nanobiomarkers

Despite recent advances, patients with malignant brain tumors still have a poor prognosis. Glioblastoma (WHO grade 4 astrocytoma), the most malignant brain tumor, represents 50% of all astrocytomas, with a median survival rate of <1 year. It is, therefore, extremely important to search for new diagnostic and therapeutic approaches for patients with glioblastoma. This study describes the application of superparamagnetic nano-particles of iron oxide, as well as monoclonal antibodies, of immunophenotypic significance, conjoined to quantum dots for the ultrastructural assessment of glioblastoma cells. For this proposal, an immunophenotypic study by flow cytometry was carried out, followed by transmission electron microscopy analysis. The process of tumor cell labeling using nanoparticles can successfully contribute to the identification of tumorigenic cells and consequently for better understanding of glioblastoma genesis and recurrence. In addition, this method may help further studies in tumor imaging, diagnosis, and prognostic markers detection.

Effects of Spider Venom Toxin PWTX-I (6-Hydroxytrypargine) on the Central Nervous System of Rats

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

Subcellular localization of proteins labeled with GFP in Xanthomonas citri ssp citri: targeting the division septum

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

Expression of Ki-67 Antigen and Caspase-3 Protein in Benign Lesions and Esophageal Carcinoma

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