Rogéria Serakides

Osteogenic differentiation of mesenchymal stem cells from osteopenic rats subjected to physical activity with and without nitric oxide synthase inhibition

Physical activity has potent and complex effects on bones. We hypothesized that physical activity has a positive effect upon osteopenic rat bones because it stimulates osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We also postulated that local nitric oxide concentrations mediate the effects of physical activity on bones. The objective of this study was to investigate the osteogenic differentiation in vitro of MSCs from osteopenic female rats subjected to physical activity with and without nitric oxide synthase inhibition. We used MSCs from the femurs of Wistar female rats divided into six groups: Group 1, sham-operated (control); Group 2, sedentary osteopenic; Group 3, active osteopenic; Group 4, sham-operated with L-NAME; Group 5, sedentary osteopenic with L-NAME; and Group 6, active osteopenic with L-NAME. The cells were cultured at 37 degrees C and 5% CO2. Cells were phenotypically characterized with anti-CD45, anti-CD90, anti-CD73, and anti-CD54 using a FACScan cytometer. MSCs were cultured in osteogenic medium for 7, 14 and 21 days. Alkaline phosphatase activity, the capacity of dimethylthiazol conversion in formazan crystals, collagen synthesis and the number of mineralized nodules were analyzed. The means of all of the variables were compared using the SNK test. MSCs did not express CD45 in 96.94% of the cells, but there was expression of CD73, CD54 and CD90 in 93.99%, 95.10% and 86.77% of the cells, respectively. MSCs from osteopenic rats showed less osteogenic differentiation. Surprisingly, physical activity increased the osteogenic differentiation of MSCs in osteopenic rats. Inhibition of nitric oxide synthase in vivo had a negative effect upon the osteogenic potential of MSCs from normal rats and from osteopenic rats subjected to physical activity. Our results suggest that nitric oxide stimulates MSCs osteogenic differentiation and that nitric oxide mediates the beneficial effects of physical activity upon MSCs osteogenic differentiation.

IMMUNOPHENOTYPIC FEATURES OF TUMOR INFILTRATING LYMPHOCYTES FROM MAMMARY CARCINOMAS IN FEMALE DOGS ASSOCIATED WITH PROGNOSTIC FACTORS AND SURVIVAL RATES

BackgroundThe immune system plays an important role in the multifactorial biologic system during the development of neoplasias. However, the involvement of the inflammatory response in the promotion/control of malignant cells is still controversial, and the cell subsets and the mechanisms involved are poorly investigated. The goal of this study was to characterize the clinical-pathological status and the immunophenotyping profile of tumor infiltrating lymphocytes and their association with the animal survival rates in canine mammary carcinomas.MethodsFifty-one animals with mammary carcinomas, classified as carcinomas in mixed tumors-MC-BMT = 31 and carcinomas-MC = 20 were submitted to systematic clinical-pathological analysis (tumor size; presence of lymph node and pulmonary metastasis; clinical stage; histological grade; inflammatory distribution and intensity as well as the lymphocytic infiltrate intensity) and survival rates. Twenty-four animals (MC-BMT = 16 and MC = 8) were elected to the immunophenotypic study performed by flow cytometry.ResultsData analysis demonstrated that clinical stage II-IV and histological grade was I more frequent in MC-BMT as compared to MC. Univariate analysis demonstrated that the intensity of inflammation (moderate/intense) and the proportion of CD4+ (≥ 66.7%) or CD8+ T-cells (<33.3%) were not associated with worse survival rate. Multivariate analysis demonstrated that only lymphocytic infiltrate intensity ≥ 600 (P = 0.02) remained as independent prognostic factor. Despite the clinical manifestation, the lymphocytes represented the predominant cell type in the tumor infiltrate. The percentage of T-cells was higher in animals with MC-BMT without metastasis, while the percentage of B-lymphocytes was greater in animals with metastasized MC-BMT (P < 0.05). The relative percentage of CD4+ T-cells was significantly greater in metastasized tumors (both MC-BMT and MC), (P < 0.05) while the proportion of CD8+ T-cells was higher in MC-BMT without metastasis. Consequently, the CD4+/CD8+ ratio was significantly increased in both groups with metastasis. Regardless of the tumor type, the animals with high proportions of CD4+ and low CD8+ T-cells had decreased survival rates.ConclusionThe intensity of lymphocytic infiltrate and probably the relative abundance of the CD4+ and CD8+ T-lymphocytes may represent important survival prognostic biomarkers for canine mammary carcinomas.

Intra-bone marrow injection of mesenchymal stem cells improves the femur bone mass of osteoporotic female rats

The effect of intra-bone injection of differentiated rat bone marrow mesenchymal stem cells (BMMSCs) into the femur of osteoporotic female rats was studied. Osteoporosis was induced in Wistar female rats by bilateral ovariectomy. Then, 0.75 million BMMSCs isolated from healthy rats were injected into the femurs of osteoporotic rats. Histomorphometric analysis and histology clearly revealed improvements in the treated group as compared to untreated group. In 2 months, the femurs of treated rats, unlike untreated rats, showed trabecular bone percentage almost similar to the femurs from control healthy rats. To confirm the origin of newly formed bone, the experiment was repeated with BMMSCs isolated from green fluorescent protein transgenic rats. Confocal microscopy demonstrated green fluorescent protein-positive cells at the surface of trabecular bone of the treated rats. We investigated in vitro osteogenic differentiation of BMMSCs isolated from osteoporotic rats by studying alkaline phosphatase activity, collagen synthesis, and the ability to form mineralized nodules. Osteoporotic BMMSCs showed less differentiation capabilities as compared to those isolated from healthy rats. The results clearly demonstrated the importance of BMMSCs in osteoporosis and that the disease can be treated by injection of BMMSCs.

Protective immunity induced by rPb27 of Paracoccidioides brasiliensis

A cDNA coding for an antigenic protein (rPb27) from the pathogenic fungus Paracoccidioides brasiliensis was cloned and its protective activity was determined against paracoccidioidomycosis (PCM). The cDNA sequence contained an open reading frame (ORF) of 660 base pairs encoding a protein of 219 amino acids with a predicted molecular weight of 25kDa. The deduced amino acid sequence exhibited 100% identity to the 27kDa P. brasiliensis hypothetic protein (access number AA49615). The complete coding cDNA was cloned into a pGEX 4T-2 plasmid and expressed in Escherichia coli as a glutathione-S-transferase-tagged (GST) recombinant protein. Mice immunized with purified rPb27 were able to develop high levels of IgG2b, moderate levels of IgG1 and low levels of IgG2a. At the same time the levels of TGF-beta and IFN-gamma were high while a very low production of IL-10 was verified. Using confocal microscopy with anti-rPb27 mouse serum against P. brasiliensis yeast forms, surface and cytosolic staining pattern were observed. Moreover, immunization of mice with this antigen induced a significant degree of protection in the lungs (93%), liver (93%) and spleen (100%) at 60 days after challenge with infection. Thus, the granulomatous lesions revealed a greater degree of compaction and organization, with few lesions in the lungs and no dissemination of the fungus to other organs. These results showed that a recombinant protein of P. brasiliensis (rPb27) promoted acquired protection against infection with P. brasiliensis yeast forms, suggesting the use of this protein for future development as a prophylactic vaccine for PCM.

In vitro and in vivo osteogenic potential of bioactive glass-PVA hybrid scaffolds colonized by mesenchymal stem cells

Bioactive glass/polymer composites are promising materials for bone tissue engineering. The present research group has developed porous hybrid scaffolds comprised of 50% polyvinyl alcohol/50% bioactive glass with a 70%SiO(2)-30%CaO composition. Prior studies have also shown the adequate structural and mechanical behavior of these scaffolds. As such, the present study investigates the in vitro and in vivo osteogenic potential of the scaffold, using mesenchymal stem cells (MSC) from the bone marrow of female rats. MTT, alkaline phosphatase activity, collagen secretion and Von Kossa staining were conducted to evaluate the differentiation ability of MSC in an osteogenic medium. The in vitro results indicate an increase in both cell proliferation and osteogenic differentiation when the hybrid material is present. Von Kossa staining showed a progressive increase in mineralization nodules, coupled with time differentiation. For the in vivo evaluation, three groups were studied: (1) group implanted with the hybrid scaffold, (2) group implanted with scaffold colonized by non-differentiated MSC and (3) group implanted with scaffold colonized by differentiated MSC. The scaffolds were subcutaneously implanted on the back of Wistar rats for 1-8 weeks, and histological and histomorphometric analyses were performed. The tissue ingrowth proved to be higher in the groups colonized by MSC in the first week. In the second week, only the hybrid colonized by differentiated MSC presented a larger percentage of connective tissue. In the third, fourth and eighth weeks, all groups presented 70% of the hybrid scaffold filled with tissue. However, only the group with differentiated MSC presented some form of osteoid tissue, indicating that the hybrid scaffold with differentiated MSC does indeed present osteogenic potential.

Effect of growth hormone and induced IGF-I release on germ cell population and apoptosis in the bovine testis

Bovine growth hormone has been used in dairy cattle to increase milk production,but it also increases the twin parturition rate. This effect is mediated by insulin-like growth factor-I (IGF-I), which prevents follicular atresia by hindering apoptosis of granulosa cells. The action of GH and IGF-I on testicular function remains unclear. The goal of this study, therefore, was to verify the effects of short-term administration of GH and induced IGF-I release on the number of testicular germ cells, testicular morphology, and apoptosis in the bovine testis. Twenty Zebu bulls were split into 2 groups. The bulls in Group 1 (n = 10) were treated with 2 subcutaneous injections of bovine GH (500 mg/bull) 7 d apart. Group 2 bulls (n = 10) received placebos under the same protocol. All of the bulls were slaughtered 14 d after the start of treatment. Fragments of the testis were collected, fixed in Bouins solution, embedded in paraffin, and the sections stained with hematoxilin and eosin. The paraffin-embedded sections were also used for in situ detection of apoptotic cells. Blood samples were collected at slaughter to measure serum levels of IGF-I, FSH and LH. Neither the number of Stage I seminiferous epithelium germ cells and the morphometric parameters (tubular diameter, seminiferous epithelium height, and volumetric proportions of structural components) nor the blood levels of FSH and LH showed a significant difference between the 2 groups. However, the treated animals showed an increase in serum IGF-I (P<0.01). Apoptotic germ cells were detected in the testis of both groups, showing the same pattern and a stage-specific apoptosis pattern. Most of the labeled cells were spermatocytes. The localization of apoptotic germ cells did not differ between groups. These results suggest that short-term administration of GH does not affect bovine spermatogenesis in adult bulls.

Carcinogênese hormonal e neoplasias hormônio-dependentes

The hormone-dependent neoplasms may originate from genetic mutations resulting the normal cells proliferation or of the cells multiplication already transformed by other carcinogen. Some hormones and anti-hormones drugs are being used with success in the treatment of those neoplasm in humans. In spite of the elevated frequency of the hormone dependent neoplasms in the animals, the use of antagonistic hormones and anti-hormones drugs, as well as the identification immunohistochemical of hormones receptors in the neoplasm is not already part of the veterinary hospitals routine.

In vivo evaluation of bioactive glass foams associated with platelet-rich plasma in bone defects.

The objective of this study was to evaluate the use of bioactive glass foams produced by the sol–gel process, associated or not with platelet‐rich plasma (PRP), in the regeneration of bone defects. Mongrel dogs (n = 14) were divided into two groups after having their superior first premolar removed. A small piece of vestibular bone from the alveolus was intentionally removed. The area was filled with bioactive glass foam produced by the sol–gel method. Two groups were tested: group A was the glass foam; group B was the same material associated with PRP, prepared from each animal. The other side of alveolar bone was used as a control group, in which the bone defect did not receive any biomaterial. The thickness of the bone area was measured before and after the intervention. After a period of 60 days implantation, the right and left bones were measured again, and a bone biopsy on both regions was conducted for histological analysis. The findings show an increase of bone thickness for both materials implanted compared to the control group. Group B, implanted with bioactive glass foam associated with PRP, showed a thicker bone area compared to Group A. Histological results indicate bone formation for both materials used. However, the bioactive glass associated with PRP gave rise to a more mature bone formation. These results show that bioactive glass foams processed by a sol–gel method is effective in maintaining the thickness of the alveolar ridge, and the use of PRP associated with the foams improve bone formation. Copyright

Foliculogênese e esteroidogênese ovarianas em ratas adultas hipertireóideas

Ovarian folliculogenesis and steroidogenesis were studied in hyperthyroid adult rats. Hyperthyroidism was induced in 27 five month-old Wistar rats by daily administration of 50µg L-thyroxin. Other 27 rats were maintained in euthyroid state as controls. At 30, 60 and 90 days after the beginning of the treatment, nine rats from each group were sacrificed. The ovaries were weighed, inspected and processed for histomorphometric evaluation. Plasmatic levels of free T4, estradiol and progesterone were determined. The levels of T4 were significantly higher in the hyperthyroid rats at 30, 60 and 90 days, but significant differences on the ovarian weight were detected only at 90 days. The number of secondary and tertiary follicles and of corpus luteum was greater at 60 or 90 days, but the rate of follicular atresia was different only at 90 days. No significant differences were observed on the number of primary and pre-ovulatory follicles and on the concentrations of estradiol and progesterone. Our data suggest that hyperthyroidism stimulates ovarian folliculogenesis in sexually mature rats. Furthermore, there is a reduction on the rate of follicular atresia.

Relação tireóide-gônadas e níveis plasmáticos de fósforo, cálcio e fosfatase alcalina em ratas

The interrelation between thyroid, gonads and osseous metabolism was studied in either intact or castrated adult female rats kept under hyperthyroidism or euthyroidism for 30, 60, or 90 days. Plasmatic levels of phosphorus, calcium, and alkaline phosphatase were measured to assess the osseous metabolism. These characteristics were correlated to the levels of estrogen, progesterone, and free T4. Either hypogonadism or hyperthyroidism interfered with the plasmatic characteristics of osseous metabolism. Hyperphosphatemia and hypocalcemia were induced by hyperthyroidism, whereas the hypogonadism had little effect on the levels of phosphorus, but it had a potencialization effect on the hyperphosphatemia and hypocalcemia induced by hyperthyroidism. The effect of hypogonadism on the alkaline phosphatase activity was reduced by the hyperthyroidism.