MC3T3-E1 Cells Behavior on Surfaces Bombarded by Argon Ions in Planar Cathode Discharge.

To evaluate the effect of topography in nanoscale, titanium surfaces were bombarded by argon ions (a chemically inert gas), in an atmosphere of plasma. The effects of surface parameters on morphology, adhesion, proliferation, and MC3T3-E1 preosteoblasts differentiation were analyzed. Nontreated (smooth) surfaces were used as a control. The levels of average roughness (Ra) observed in bombarded and smooth titanium surfaces were of 95 and 14 nm, respectively. The wettability increased on treated surfaces. The number of attached cells (30 and 60 min) was significantly higher on the bombarded surface. The cell proliferation after 3 and 7 days was also significantly higher on the ion-bombarded surface. In addition, the ALP activity and expression of osteocalcin were higher in cells grown on the treated surface. The results showed that bombardment with argon ions increased the roughness and the wettability of the Ti surface, promoting a significant increase in the adhesion, proliferation, and differentiation of preosteoblasts.

Analysis of HFE genes C282Y, H63D, and S65D in patients with hyperferritinemia from northeastern Brazil.

BACKGROUND: Hereditary hemochromatosis (HH) is a genetic disease caused by the high absorption and deposition of iron in several organs. This accumulation results in several clinical complications such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders, and skin darkening. The H63D and C282Y mutations are well defined in the HH etiology. The objective of this article is identification of the H63D and C282Y mutations in the HFE protein gene and the frequency assessment of these mutations in patients with persistent increase of serum ferritin in patients from Natal City from state of Rio Grande do Norte, located in northeastern Brazil. RESULTS: Of the 299 patients studied for C282Y and H63D, 48.49% showed absence of mutation and 51.51% showed some sort of mutation: heterozygous C282Y mutation in 4.35% patients, homozygous C282Y mutation in 2.67% patients, heterozygous H63D mutation in 31.44% patients, homozygous H63D mutation in 8.03% patients, and heterozygous for the mutation in both genes (C282Y/H63D) in 5.02% patients. The S65C mutation was studied in 112 patients and heterozygous mutation (S65D/WT) in 2.67% of patients and double mutation (H63D/S65C) in 1.78% of patients were observed. CONCLUSION: Due to the high prevalence of hemochromatosis, its genetic diagnosis has become a challenge, especially in the high-risk group.

Coexpression of p53 protein and MDR functional phenotype in leukemias: the predominant association in chronic myeloid leukemia.

BACKGROUND: One of the best characterized resistance mechanisms of leukemias is multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) and multidrug-resistant related protein (MRP). In addition to Pgp and MRP, p53 mutation or inactivation might play a relevant role in therapeutic failure. Some studies have demonstrated that Pgp and MRP may be activated in association with overexpression of mutant or inactivated p53 protein. The aim of this study was to investigate the association between p53 expression and MDR functional phenotype analyzed by flow cytometry (FCM). METHODS: Rhodamine-123 assay analyzed by FCM was used to detect the MDR phenotype that was positive in 18 out of 41 (43.9%) cases of chronic myeloid leukemia (CML), 16 out of 28 (57.1%) chronic lymphoid leukemia (CLL) cases, 11 out of 28 (39.3%) acute myeloid leukemia (AML) cases, and four out of 22 (18.2%) acute lymphoid leukemia (ALL) cases. RESULTS: Variable levels of p53 expression were observed in leukemic cells: 12 out of 41 (29.2%) in CML, nine out of 28 (32.1%) in CLL, 15 out of 28 (53.6%) in AML, and eight out of 22 (36.4%) in ALL samples. CONCLUSIONS: In our study, no significant association between p53 expression and MDR functional phenotype was observed in ALL, CLL, and AML. On the other hand, a significant association (P = 0.0003) of the coexpression was observed in CML. The p53 overexpression was more frequently seen in the accelerated phase and the blastic phase of this disease. Our results suggest that an MDR functional phenotype could be associated with p53 mutation in the advanced stage of leukemias.

Cytokinesis-block micronucleus assay adapted for analyzing genomic instability of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are multipotent cells used in cell therapy research. One of the problems involving hMSCs is the possibility of genetic instability during in vitro expansion required to obtain a suitable number of cells for clinical applications. The cytokinesis-block micronucleus (CBMN) assay measures genetic instability by analyzing the presence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) in binucleated cells. The present study describes modifications in the CBMN assay methodology to analyze genetic instability in hMSCs isolated from the umbilical vein and in vitro expanded. The best protocol to achieve binucleated hMSCs with preserved cytoplasm was as follows: cytochalasin B concentration (4.0 µg/mL), use of hypotonic treatment (3 min), and the fixative solution (9 methanol:1 acetic acid). These adaptations were reproduced in three hMSC primary cell cultures and also in XP4PA and A549 cell lines. The frequency of hMSCs treated with mitomycin-C presenting MN was lower than that with other nuclear alterations, indicating that the hMSCs contain mechanisms to avoid a high level of chromosomal breaks. However, a high frequency of cells with NPBs was detected and spontaneous anaphase bridges under normal hMSC in vitro culture were observed. Considering that anaphase bridges are characteristic alterations in tumor cells, the CBMN assay is indicated as an important tool associated with other genetic analyses in order to ensure the safe clinical use of hMSCs in cell therapy.

Successful screening for Gaucher disease in a high-prevalence population in tabuleiro do Norte (northeastern Brazil): a cross-sectional study.

BACKGROUND: Gaucher disease (GD) is a hereditary lysosomal storage disorder characterized by the accumulation of glucosylceramide, mainly in the cells of the reticuloendothelial system, due to a deficiency of the enzyme acid ß-glucosidase (GBA). Diagnosis is usually based on measurement of GBA activity in peripheral leukocytes. The purpose of this study was to evaluate the ability of screening for GBA and chitotriosidase activity using dried blood spots on filter paper (DBS-FP) to identify individuals at high risk for GD in high-risk populations such as that of Tabuleiro do Norte, a small town in Northeastern Brazil. METHODS: Between 1 June 2007 and 31 May 2008, 740 consented residents and descendants of traditional families from Tabuleiro do Norte were submitted to screening with DBS-FP. Subjects with GBA activity < 2.19 nmol/h/mL were referred to the analysis of GBA and chitotriosidase activity in peripheral leukocytes and in plasma, respectively. Subjects at highest risk for GD (GBA activity in peripheral leukocytes < 5.6 nmol/h/mg protein) were referred to molecular analysis to confirm diagnosis. RESULTS: Screening with DBS-FP identified 135 subjects (18.2%) with GBA activity < 2.19 nmol/h/mL, 131 of whom remained in the study. In ten of these (7.6%), GBA activity in leukocytes was 2.6-5.5 nmol/h/mg protein. Subsequent molecular analysis confirmed six cases of heterozygosity and four normals for GD. CONCLUSION: DBS-FP assay was shown to be an effective initial GD-screening strategy for high-prevalence populations in developing regions. Diagnosis could not be established from GBA activity in leukocytes alone, but required confirmation with molecular analysis.

p53 flow cytometry evaluation in leukemias: correlation to factors affecting clinical outcome.

p53 is a cell cycle checkpoint control protein that assesses DNA damage and acts as a transcription factor regulating genes, which control cell growth, DNA repair, and apoptosis. p53 mutations have been found in a wide variety of different cancers including flow cytometric assessment of p53 protein expression using anti-p53 monoclonal antibodies. We studied p53 protein expression by flow cytometry (FC) assay in 223 blood and/or bone marrow samples from 72 patients with chronic myeloid leukemia (CML): 54 in chronic phase (CML-CP), 7 in accelerated phase (CML-AP), and 11 in blastic phase (CML-BP); 64 patients with chronic lymphoid leukemia (CLL): (34 at diagnosis, 21 in previously treated, and 9 with Richter's syndrome); 44 patients with acute lymphoid leukemia (ALL): 36 at diagnosis and 8 in relapse; and 43 acute myeloid leukemia (AML): 27 de novo, 7 in relapse, and 9 secondary. p53 protein expression was observed in 64 of 223 patient's samples: 14/64 (21.9%) CLL, 13/44 (29.5%) ALL, 19/43 (44.2%) AML, and 17/72 (23.6%) CML. Highest levels were detected in the advanced phases of CLL, ALL, and CML. In addition, in patients with AML, high levels of p53 expression were detected in secondary and relapse disease and also in de novo AML cases. Our results demonstrated that p53 expression levels are strongly associated with advanced disease. On the basis of these results, we concluded that FC can be a reliable approach to study p53 protein expression in leukemic patients.