Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

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.

Effects of ascorbic acid on in vitro culture of bovine preantral follicles

The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles. © 2012 Copyright Cambridge University Press.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos

Summary Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA. © Cambridge University Press 2011.

Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research. © 2013 Spandidos Publications Ltd. All rights reserved.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Photodynamic inactivation of Streptococcus mutans and Streptococcus sanguinis biofilms in vitro

The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using erythrosine (ER) and Rose Bengal (RB) photosensitizers and a blue light-emitting diode (LED) on the viability of Streptococcus mutans and Streptococcus sanguinis biofilms. Biofilms were grown in acrylic disks immersed in broth to production of biofilms, inoculated with microbial suspension (106 cells/mL) and incubated for 48 h. After the formation of biofilms, the effects of the photosensitizers ER and RB at a concentration of 5 μM for 5 min and blue LED (455 ± 20 nm) for 180 s, photosensitizers alone and conjugated were evaluated. Next, the disks were placed in tubes with sterile physiological solution (0.9 % sodium chloride) and sonicated for to disperse the biofilms. Tenfold serial dilutions were carried and aliquots seeded in brain heart infusion agar which were then incubated for 48 h. Then the numbers colony-forming units per milliliter (CFU/mL; log 10) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by both photosensitizers. The reductions with RB and ER were, 0.62 and 0.52 log10 CFU mL -1 for S. mutans biofilms (p = 0.001), and 0.95 and 0.88 log 10 CFU mL-1 for S. sanguinis biofilms (p = 0.001), respectively. The results showed that biofilms formed in vitro by S. mutans and S. sanguinis, were sensitive to PDI using a blue LED associated with photosensitizers ER or RB, indicating its use in the control of caries and periodontal diseases. © 2012 Springer-Verlag London Ltd.

Effect of the bone marrow cell transplantation on elevated plus-maze performance in hippocampal-injured mice

Several reports have shown that the hippocampus plays an important role in different aspects of the emotional control. There is evidence that lesions in this structure cause behavioral disinhibition, with reduction of reactions expressing fear and anxiety. Thus, to portray the aptitude of cell therapy to abrogate injuries of hippocampal tissue, we examined the behavioral effects of bone marrow mononuclear cells (BMMCs) transplantation on C57BL/6 mice that had the hippocampus damaged by electrolytic lesion. For this purpose, mice received, seven days after bilateral electrolytic lesion in the dorsal hippocampus, culture medium or BMMCs expressing the enhanced green fluorescent protein (EGFP) transgene. One week after transplantation, animals were tested in the elevated plus-maze (EPM). On the whole, three assessment sessions in the EPM were carried out, with seven days separating each trial. Thirty-five days after the induction of injury, mice were sacrificed and their brains removed for immunohistochemistry. The behavioral evaluation showed that the hippocampal lesion caused disinhibition, an effect which was slightly lessened, from the second EPM test, in transplanted subjects. On the other hand, immunohistochemical data revealed an insignificant presence of EGFP+ cells inside the brains of injured mice. In view of such scenario, we hypothesized that the subtle rehabilitation of the altered behavior might be a result from a paracrine effect from the transplanted cells. This might have been caused by the release of bioactive factors capable of boosting endogenous recuperative mechanisms for a partial regaining of the hippocampal functions. © 2013 Elsevier B.V.

Nitensidine A, a guanidine alkaloid from Pterogyne nitens, induces osteoclastic cell death

Nitensidine A is a guanidine alkaloid isolated from Pterogyne nitens, a common plant in South America. To gain insight into the biological activity of P. nitens-produced compounds, we examined herein their biological effects on osteoclasts, multinucleated giant cells that regulate bone metabolism by resorbing bone. Among four guanidine alkaloids (i.e., galegine, nitensidine A, pterogynidine, and pterogynine), nitensidine A and pterogynine exhibited anti-osteoclastic effects at 10 μM by reducing the number of osteoclasts on the culture plate whereas galegine and pterogynidine did not. The anti-osteoclastic activities of nitensidine A and pterogynine were exerted in a concentration-dependent manner, whereas nitensidine A exhibited an approximate threefold stronger effect than pterogynine (IC50 values: nitensidine A, 0.93 ± 0.024 μM; pterogynine, 2.7 ± 0.40 μM). In the present study, the anti-osteoclastic effects of two synthetic nitensidine A derivatives (nitensidine AT and AU) were also examined to gain insight into the structural features of nitensidine A that exert an anti-osteoclastic effect. The anti-osteoclastic effect of nitensidine A was greatly reduced by substituting the imino nitrogen atom in nitensidine A with sulfur or oxygen. According to the differences in chemical structures and anti-osteoclastic effects of the four guanidine alkaloids and the two synthetic nitensidine A derivatives, it is suggested that the number, binding site, and polymerization degree of isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their anti-osteoclastic effects. © 2013 Springer Science+Business Media Dordrecht.

Photodynamic potential of curcumin and blue LED against streptococcus mutans in a planktonic culture

Background: The photodynamic therapy (PDT) involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent or dye in the presence of oxygen for eradication of target cells. In dentistry, this therapy is used to suppress the growth of microorganisms involved directly with dental decay and periodontitis process. There are evidences that curcumin dye is able to control microbial activity when illuminated with specific wavelength. The purpose of this study was to evaluate the in vitro efficacy of PDT using curcumin dye (Cur-C) in combination with a blue LED (L) device on a planktonic model of Streptococcus mutans ( S. mutans). Methods: Suspensions (0.5mL) containing S. mutans at 1×107CFUmL-1 were prepared and divided into 4 groups: Group C-L- (control: no treatment and 1 experimental condition), Group C+L- (curcumin at 3 different concentrations: 2000; 4000 and 8000μM and 3 experimental conditions), Group C-L+ (LED at 3 different dosages: 24, 48 and 72Jcm-2 and 3 experimental conditions), and Group C+L+ (PDT group: curcumin at respective concentrations combined to LED dosages and 9 experimental conditions). Samples of each experimental condition were cultured in Petri dishes of BHI agar. Incubation in micro-aerophilia at 37°C for 48h was performed for subsequent visual counting of CFU/mL. Data were transformed into log10 and analyzed by two-way ANOVA and Tukey's test at p<0.05. Results: Group C. +. L+, in specific experimental conditions, demonstrated a log bacterial reduction 70% higher than Group C. -. L-. Both groups C. -. L+ and C. +. L- presented a slight decrease in log bacterial counting. Conclusion: This in vitro method was able to reduce the number of S. mutans in a planktonic suspension. © 2013 Elsevier B.V.

Uso do sistema de cultivo bidimensional e tridimensional para diferenciação de células-tronco espermatogoniais e indução da espermatogênese in vitro em murinos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Stem cell factor estimula células da musculatura lisa da traqueia a produzir TGF-β, FGF-2 E CCL3/MIP-1α

Pós-graduação em Ciências Fisiológicas - FOA

Atividade antibacteriana e citotoxicidade do agregado de trióxido mineral associado a diferentes concentrações de clorexidina

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Drifter technique: a new method to obtain metaphases in Hep-2 cell line cultures

A linhagem celular Hep-2 é formada por células de carcinoma da laringe e é muito utilizada em modelos de carcinogênese e mutagenêse. Para avaliar o potencial proliferativo desta linhagem, desenvolvemos uma metodologia citogenética (técnica do sobrenadante) para obtenção de metáfases a partir de células que, ao entrarem em mitose, perdem adesão celular, ficando em suspensão no meio de cultura. Através deste procedimento, foram contadas 2000 células, correspondendo a um índice mitótico (IM) de 22.2% . Apesar de o IM obtido por esta técnica não ter sido estatisticamente diferente do IM obtido por outras metodologias citogenéticas clássicas, a técnica do sobrenadante é vantajosa porque elimina o uso de alguns reagentes utilizados na obtenção de metáfases e também diminui o consumo de reagentes de manutenção desta linhagem.

Efeito do estradiol e da progesterona no desenvolvimento e na qualidade de embriões bovinos produzidos in vitro

Avaliaram-se o desenvolvimento e a qualidade de embriões bovinos, cocultivados com células epiteliais do oviduto bovino (CEOBs) expostas ou não ao estradiol e à progesterona. Os ovócitos foram maturados in vitro por 24h e, então, fertilizados utilizando-se sêmen congelado, em estufa de CO2 a 5% e 38,5oC. As CEOBs foram cultivadas em TCM-199 com ou sem estradiol (E2) (24 horas), nas mesmas condições da maturação e fertilização in vitro (MIV e FIV), e, em seguida, adicionadas aos diferentes grupos em CR2 com ou sem progesterona (P4) (G1=P4+E2); (G2=E2); (G3=P4) e (G4=controle). Após 18h da FIV, as células foram cultivadas nos diferentes sistemas. Nenhuma diferença (P>0,05) foi observada nas taxas de clivagem entre G1, G2 e G4 (53,5%; 56,3%; 51,7%) e nos padrões de blastocistos (BLs) (29,3%; 31,2%, 28,7%). Índices menores (P<0,05) foram obtidos no G3 para ambas as variáveis (34,5%; 16,4%). G1 e G2 apresentaram taxas de eclosão maiores (P<0,05) que os outros grupos (23,3%; 23,2%), sendo G4 (19,3%) diferente de G3 (16,1%). Em G1, G2 e G3, o número de células nos BLs aumentou 125,9; 128,4 e 123,6, respectivamente (P<0,05), em relação ao G4 (112,5). Conclui-se que o tratamento das CEOBs com o E2, nas primeiras 24 horas de cultivo, pode ser usado isoladamente ou em combinação com a progesterona, a fim de melhorar a qualidade de embriões bovinos produzidos in vitro.