An. Acad. Bras. Ciênc.

Abstract Mesenchymal stem cells present clinical potential to recover and regenerate injured tissues in diverse pathologies. The in vitro expansion and characterization of these cells contribute to elucidation of the mechanisms of senescence and strategies involving cell therapies. This study aimed to compare specific characteristics between initial and advanced passages of mesenchymal stem cells derived from adipose tissue and bone marrow. Both cell types were characterized according to immunophenotype, osteogenic differentiation, genomic instability, migration assay, doubling population time and colony forming ability. Our results demonstrated that both cell types were able to maintain an immunophenotypic profile typical of mesenchymal stem cells during increasing passages. Adipose stem cells at initial passage presented greater migration capacity compared to advanced passage cells, and advanced passage cells proliferated faster than initial passage cells. Bone marrow stem cells at early passages presented higher osteogenic potential than advanced. At advanced passages they presented higher colony forming capacity and genetic damage than those at initial passage. These results suggest that mesenchymal stem cells maintained in culture presented characteristics of senescence that should be monitored prior the use in regenerative medicine and cells derived from bone marrow at initial passage have better potential for therapeutic use in bone tissue engineering

Analysis of mRNA transcripts in chronic myeloid leukemia patients

The nature of BCR/ABL hybrid mRNA was analyzed by RT-PCR in cells from 33 patients (22 males, 11 females) with chronic myeloid leukemia (CML). b3a2 mRNA was found in 14 cases, whereas 13 patients had b2a2 mRNA and six had both kinds of mRNA, with a predominance of the b3a2 type. The type of mRNA present showed no significant correlation with age, hemoglobin level, number of leukocytes and platelets, percentage of blasts or basophils or the presence of splenomegaly at diagnosis. There was also no correlation with sex or duration of the chronic phase. When these results were combined with those reported by other groups, a significant association (P = 0.029) was observed for mRNA type vs. sex, with a predominance of men in the groups expressing b2a2 (2.68:1) and b3a2 (1.33:1). We conclude that the classification of patients according to mRNA type does not homogenize the clinical and hematological data within groups, where variance is large, nor does it allow a differentiation between groups

Gemifloxacin mesylate (GFM) stability evaluation applying a validated bioassay method and in vitro cytotoxic study

AbstractThe validation of a microbiological assay applying the cylinder–plate method to determine the quinolone gemifloxacin mesylate (GFM) content is described. Using a strain of Staphylococcus epidermidis ATCC 12228 as the test organism, the GFM content in tablets at concentrations ranging from 0.5 to 4.5μgmL−1 could be determined. A standard curve was obtained by plotting three values derived from the diameters of the growth inhibition zone. A prospective validation showed that the method developed is linear (r=0.9966), precise (repeatability and intermediate precision), accurate (100.63%), specific and robust. GFM solutions (from the drug product) exposed to direct UVA radiation (352nm), alkaline hydrolysis, acid hydrolysis, thermal stress, hydrogen peroxide causing oxidation, and a synthetic impurity were used to evaluate the specificity of the bioassay. The bioassay and the previously validated high performance liquid chromatographic (HPLC) method were compared using Student's t test, which indicated that there was no statistically significant difference between these two validated methods. These studies demonstrate the validity of the proposed bioassay, which allows reliable quantification of GFM in tablets and can be used as a useful alternative methodology for GFM analysis in stability studies and routine quality control. The GFM reference standard (RS), photodegraded GFM RS, and synthetic impurity samples were also studied in order to determine the preliminary in vitro cytotoxicity to peripheral blood mononuclear cells. The results indicated that the GFM RS and photodegraded GFM RS were potentially more cytotoxic than the synthetic impurity under the conditions of analysis applied

Experimental Model of Gene Transfection in Healthy Canine Myocardium: Perspectives of Gene Therapy for Ischemic Heart Disease

OBJECTIVE: To assess the transfection of the gene that encodes green fluorescent protein (GFP) through direct intramyocardial injection. METHODS: The pREGFP plasmid vector was used. The EGFP gene was inserted downstream from the constitutive promoter of the Rous sarcoma virus. Five male dogs were used (mean weight 13.5 kg), in which 0.5 mL of saline solution (n=1) or 0.5 mL of plasmid solution containing 0.5 µg of pREGFP/dog (n=4) were injected into the myocardium of the left ventricular lateral wall. The dogs were euthanized 1 week later, and cardiac biopsies were obtained. RESULTS: Fluorescence microscopy showed differences between the cells transfected and not transfected with pREGFP plasmid. Mild fluorescence was observed in the cardiac fibers that received saline solution; however, the myocardial cells transfected with pREGFP had overt EGFP expression. CONCLUSION: Transfection with the EGFP gene in healthy canine myocardium was effective. The reproduction of this efficacy using vascular endothelial growth factor (VEGF) instead of EGFP aims at developing gene therapy for ischemic heart disease

Direct intramyocardial transthoracic transplantation of bone marrow mononuclear cells for non-ischemic dilated cardiomyopathy: INTRACELL, a prospective randomized controlled trial

Objective: We tested the hypothesis that direct intramyocardial injection of bone marrow mononuclear cells in patients with non-ischemic dilated cardiomyopathy can improve left ventricular function and physical capacity. Methods: Thirty non-ischemic dilated cardiomyopathy patients with left ventricular ejection fraction <35% were randomized at a 1:2 ratio into two groups, control and treated. The bone marrow mononuclear cells group received 1.06&#177;108 bone marrow mononuclear cells through mini-thoracotomy. There was no intervention in the control group. Assessment was carried out through clinical evaluations as well as a 6-min walk test, nuclear magnectic resonance imaging and echocardiogram. Results: The bone marrow mononuclear cells group showed a trend toward left ventricular ejection fraction improvement, with magnectic resonance imaging - at 3 months, showing an increase from 27.80&#177;6.86% to 30.13&#177;9.06% (P=0.08) and returning to baseline at 9 months (28.78%, P=0.77). Magnectic resonance imaging showed no changes in left ventricular ejection fraction during follow-up of the control group (28.00&#177;4.32%, 27.42&#177;7.41%, and 29.57&#177;4.50%). Echocardiogram showed left ventricular ejection fraction improved in the bone marrow mononuclear cells group at 3 months, 25.09&#177;3.98 to 30.94&#177;9.16 (P=0.01), and one year, 30.07&#177;7.25% (P=0.001). The control group showed no change (26.1&#177;4.4 vs 26.5&#177;4.7 and 30.2&#177;7.39%, P=0.25 and 0.10, respectively). Bone marrow mononuclear cells group showed improvement in New York Heart Association functional class, from 3.40&#177;0.50 to 2.41&#177;0.79 (P=0.002); patients in the control group showed no change (3.37&#177;0.51 to 2.71&#177;0.95; P=0.17). Six-minute walk test improved in the bone marrow mononuclear cells group (348.00&#177;93.51m at baseline to 370.41&#177;91.56m at 12 months, P=0.66) and there was a non-significant decline in the control group (361.25&#177;90.78m to 330.00&#177;123.42m after 12 months, P=0.66). Group comparisons were non-significant. Conclusion: The trend of intragroup functional and subjective improvement was not confirmed when compared to the control group. Direct intramyocardial application of bone marrow mononuclear cells in non-ischemic dilated cardiomyopathy was not associated with significant changes in left ventricular function. Differences observed within the bone marrow mononuclear cells group could be due to placebo effect or low statistical power