Nuclear phenotypes and morphometry of human secretory prostate cells: A comparative study of benign and malignant lesions in Brazilian patients

Prostatic lesions in Brazilian patients with benign prostatic hyperplasia (BPH, 26 cases) or adenocarcinoma (AC, 25 cases) were compared by qualitative microscopy and morphometric analysis. In 12 cases of BPH, prostate regions with no histological alterations were considered as controls (Ct). Archival material consisted of formalin-fixed, paraffin-embedded specimens obtained from prostatic transurethral resection and radical prostatectomy. Haematoxylin/eosin (HE)-stained sections were used to estimate the nuclear areas, perimeters and form factor values. HE-stained sections from AC specimens were also used for Gleason grading. BPH, AC and Ct could be discriminated by their nuclear areas and nuclear perimeters, but not by the nuclear form factor parameter. No significant differences were found when the AC data were compared using the combined version or the predominant grade version of the Gleason score (p = 0.8380 for nuclear area; p = 0.6076 for nuclear perimeter; p = 0.9202 for nuclear form factor; n = 200 nuclei per patient). This finding indicates that there is extensive heterogeneity in the size and shape of the nucleus in AC cells. These results also show that although the nuclear morphometry served to discriminate BPH and AC from each other and from Ct, it was not sufficient to correlate AC lesions with their respective Gleason scores in the human population analyzed.

Paracoccidioidomycosis: No genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay

Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis. As infectious diseases can cause DNA damage, the authors aimed at analyzing DNA breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. The results suggested that paracoccidioidomycosis does not cause genotoxicity.

Effects of hemostatic agents on the histomorphologic response of human dental pulp capped with calcium hydroxide

Objective: To evaluate the response of human pulps capped with a calcium hydroxide [Ca(OH)2] cement after bleeding control with 2 hemostatic agents. Method and Materials: Pulps were exposed on the occlusal floor, and the bleeding was controlled either with saline solution (SS) or 2.5% sodium hypochlorite (NaOCI) (SH). After that, the pulp was capped with Ca(OH) 2 cement and restored with resin composite. After 30 (groups SS30 and SH30) and 60 (groups SS60 and SH60) days, the teeth were extracted and processed with hematoxylin-eosin and categorized in a histologic score system. The data were subjected to Kruskal-Wallis and Mann-Whitney tests (α = .05). Results: Regarding dentin bridge formation, an inferior response of SH60 group was observed when compared to SS60 (P < .05). The response of the SH30 group generally was similar to that of the groups treated with saline solution. However, after 60 days, 2.5% NaOCl showed a trend toward having an inferior response. Conclusion: Using saline solution as a hemostatic agent before pulp capping with Ca(OH)2 resulted in a significantly better histomorphologic response than using 2.5% NaOCl as a hemostatic agent before capping with Ca(OH)2.

Effect of time in hardness test on artificially demineralized human dental enamel

A cross-sectional microhardness (CSMH) test was carried out in human dental enamel exposed to a demineralizing solution in order to evaluate two different times of indentation in sound tissue and artificially induced caries. Twenty caries-free extracted human molars had one of their smooth surfaces sectioned and the enamel surface was isolated with nail polish except for an area of 6 mm2. These specimens were submitted to artificially induced enamel caries on a lactate buffer containing 0.1 ppm fluoride (F) during 28 days. All specimens were bisected to create groups A and B in which CSMH test was performed employing a Knoop indenter with a 25g load for 5 or 10 s, respectively. Student's paired t-test (p<0.05) was used to determine statistically significant differences between group A and B in 7 depths. There were no significant differences between any of the analyzed depths. Since the present experiment showed no significant difference when comparing indentations made with a 25 g load during either 5 or 10 s in different depths, this method can be used with either one of the time intervals tested without compromising a CSMH test on artificially demineralized human enamel.

Response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent

Aim: To evaluate the treatment with corticosteroid/antibiotic dressing in pulpotomy with calcium hydroxide. Methods: Forty-six premolars were pulpotomized and randomly assigned into 3 groups. In Group I pulpal wound was directly capped with calcium hydroxide, and Group II and Group III received corticosteroid/antibiotic dressing for 10 min or 48 h, respectively, before pulp capping. Teeth were processed for histological analysis after 7, 30 or 60 days to determine inflammatory cell response, tissue disorganization, dentin bridge formation and presence of bacteria. Attributed scores were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). Results: On the 7th day, all groups exhibited dilated and congested blood vessels in the tissue adjacent to pulpal wound. The inflammatory cell response was significantly greater in Group III (p<0.05). On the 30th day, in all groups, a thin dentin matrix layer was deposited adjacent to the pulpal wound and a continuous odontoblast-like cell layer underlying the dentin matrix was observed. On the 60th day, all groups presented a thick hard barrier characterized by an outer zone of dystrophic calcification and an inner zone of tubular dentin matrix underlined by a defined odontoblast-like cell layer. Conclusions: Within the limitations of present study, considering that the treatment was performed in healthy teeth, it may be concluded that the use of a corticosteroid/antibiotic dressing before remaining tissue protection with calcium hydroxide had no influence on pulp tissue healing.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers-alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor)-and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus. © 2012 S. Karger AG, Basel.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

Immunophenotypic, immunocytochemistry, ultrastructural, and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n=64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. © 2013 Wiley Periodicals, Inc.

Equine tendonitis therapy using mesenchymal stem cells and platelet concentrates: A randomized controlled trial

Introduction. Tendon injury is a major cause of lameness and decreased performance in athletic equines. Various therapies for tendonitis have been described; however, none of these therapies results in complete tissue regeneration, and the injury recurrence rate is high even after long recovery periods involving rest and physiotherapy. Methods. A lesion was induced with collagenase gel in the superficial digital flexor tendon in the center portion of the metacarpal region of eight equines of mixed breed. After two weeks, the lesions of the animals in the treated and control groups were treated through the intralesional administration of mesenchymal stem cells derived from adipose tissue (adMSCs) suspended in platelet concentrate (PC) and with phosphate buffered saline (PBS), respectively. Serial ultrasound analyses were performed every two weeks. After 16 weeks of therapy, a biopsy was performed for histopathological, immunohistochemical and gene expression (type I collagen (COL1A1), type III collagen (COL3A1), tenascin-C (TNC), tenomodulin (TNMD), and scleraxis (SCX)) analyses. Results: Differences in the ultrasound and histopathological analyses were observed between the groups. Improved results were reported in the group treated with adMSCs suspended in PC. There was no difference in the gene expression levels observed after the different treatments. The main results observed from the histopathological evaluation of the treated group were as follows: a prevention of the progression of the lesion, a greater organization of collagen fibers, and a decreased inflammatory infiltrate. A lack of progression of the lesion area and its percentage was observed in the ultrasound image, and increased blood flow was measured by Power Doppler. Conclusions: The use of adMSCs combined with PC for the therapy of experimentally induced tendonitis prevented the progression of the tendon lesion, as observed in the ultrasound examination, and resulted in a greater organization and decreased inflammation, as observed in the histopathological evaluation. These data demonstrate the therapeutic potential of this therapy for the treatment of equine tendonitis. © 2013 Carvalho et al.; licensee BioMed Central Ltd.

Interaction between mesenchymal stem cells and Ti-30Ta alloy after surface treatment

In this study, in vitro cytocompatibility was investigated in the Ti-30Ta alloy after two kinds of surfaces treatments: alkaline and biomimetic treatment. Each condition was evaluated by scanning electron microscopy/energy-dispersive X-ray spectroscopy. Cellular adhesion, viability, protein expression, morphology, and differentiation were evaluated with Bone marrow stromal cells (MSCs) to investigate the short and long-term cellular response by fluorescence microscope imaging and colorimetric assays techniques. Two treatments exhibited similar results with respect to total protein content and enzyme activity as compared with alloy without treatment. However, it was observed improved of the biomineralization, bone matrix formation, enzyme activity, and MSCs functionality after biomimetic treatment. These results indicate that the biomimetic surface treatment has a high potential for enhanced osseointegration. © 2013 Wiley Periodicals, Inc.

Citotoxicidade, genotoxicidade e expressão gênica em células da polpa dental humana sensibilizadas por cimentos endodônticos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization of mesenchymal stem cells derived from equine adipose tissue

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

Mesenchymal stem cells modulate release of matrix proteins from tendon surfaces in vitro: a potential beneficial therapeutic effect

Aim: Injury of tendons contained within a synovial environment, such as joint, bursa or tendon sheath, frequently fails to heal and releases matrix proteins into the synovial fluid, driving inflammation. This study investigated the effectiveness of cells to seal tendon surfaces and provoke matrix synthesis as a possible effective injectable therapy. Materials & methods: Equine flexor tendon explants were cultured overnight in suspensions of bone marrow and synovium-derived mesenchymal stems cells and, as controls, two sources of fibroblasts, derived from tendon and skin, which adhered to the explants. Release of the most abundant tendon extracellular matrix proteins into the media was assayed, along with specific matrix proteins synthesis by real-time PCR. Results: Release of extracellular matrix proteins was influenced by the coating cell type. Fibroblasts from skin and tendon appeared less capable of preventing the release of matrix proteins than mesenchymal stems cells. Conclusion: The source of cell is an important consideration for cell therapy.

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)