Daniela Mara de Oliveira

Differential neuroprotection by A1 receptor activation and A2A receptor inhibition following pilocarpine-induced status epilepticus

Aiming at a better understanding of the role of A(2A) in temporal lobe epilepsy (TLE), we characterized the effects of the A(2A) antagonist SCH58261 (7-(2-phenylethyl)-5-amino-2(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) on seizures and neuroprotection in the pilocarpine model. The effects of SCH58261 were further analyzed in combination with the A(1) agonist R-Pia (R(-)-N(6)-(2)-phenylisopropyl adenosine). Eight groups were studied: pilocarpine (Pilo), SCH+Pilo, R-Pia+Pilo, R-Pia+SCH+Pilo, Saline, SCH+Saline, R-Pia+Saline, and R-Pia+SCH+Saline. The administration of SCH58261, R-Pia, and R-Pia+SCH58261 prior to pilocarpine increased the latency to SE, and decreased either the incidence of or rate of mortality from SE compared with controls. Administration of R-Pia and R-Pia+SCH58261 prior to pilocarpine reduced the number of Fluoro-Jade B-stained cells in the hippocampus and piriform cortex when compared with control. This study showed that pretreatment with R-Pia and SCH58261 reduces seizure occurrence, although only R-Pia has neuroprotective properties. Further studies are needed to clarify the neuroprotective role of A(2A) in TLE.

A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) are multipotent progenitor cells used in several cell therapies. MSCs are characterized by the expression of CD73, CD90, and CD105 cell markers, and the absence of CD34, CD45, CD11a, CD19, and HLA-DR cell markers. CD90 is a glycoprotein present in the MSC membranes and also in adult cells and cancer stem cells. The role of CD90 in MSCs remains unknown. Here, we sought to analyse the role that CD90 plays in the characteristic properties of in vitro expanded human MSCs.MethodsWe investigated the function of CD90 with regard to morphology, proliferation rate, suppression of T-cell proliferation, and osteogenic/adipogenic differentiation of MSCs by reducing the expression of this marker using CD90-target small hairpin RNA lentiviral vectors.ResultsThe present study shows that a reduction in CD90 expression enhances the osteogenic and adipogenic differentiation of MSCs in vitro and, unexpectedly, causes a decrease in CD44 and CD166 expression.ConclusionOur study suggests that CD90 controls the differentiation of MSCs by acting as an obstacle in the pathway of differentiation commitment. This may be overcome in the presence of the correct differentiation stimuli, supporting the idea that CD90 level manipulation may lead to more efficient differentiation rates in vitro.

Evaluation of umbilical cord mesenchymal stem cell labeling with superparamagnetic iron oxide nanoparticles coated with dextran and complexed with Poly-L-lysine

OBJECTIVE The objective of this study was to evaluate the effect of the labeling of umbilical cord vein derived mesenchymal stem cells with superparamagnetic iron oxide nanoparticles coated with dextran and complexed to a non-viral transfector agent transfector poly-L-lysine. METHODS The labeling of mesenchymal stem cells was performed using the superparamagnetic iron oxide nanoparticles/dextran complexed and not complexed to poly-L-lysine. Superparamagnetic iron oxide nanoparticles/dextran was incubated with poly-L-lysine in an ultrasonic sonicator at 37°C for 10 minutes for complex formation superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine by electrostatic interaction. Then, the mesenchymal stem cells were incubated overnight with the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine and superparamagnetic iron oxide nanoparticles/dextran. After the incubation period the mesenchymal stem cells were evaluated by internalization of the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine and superparamagnetic iron oxide nanoparticles/dextran by Prussian Blue stain. Cellular viability of labeled mesenchymal stem cells was evaluated by cellular proliferation assay using 5,6-carboxy-fluorescein-succinimidyl ester method and apoptosis detection by Annexin V- Propidium Iodide assay. RESULTS mesenchymal stem cells labeled with superparamagnetic iron oxide nanoparticles/dextran without poly-L-lysine not internalized efficiently the superparamagnetic iron oxide nanoparticles due to its low presence detected within cells. Mesenchymal stem cells labeled with the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine efficiently internalized the superparamagnetic iron oxide nanoparticles due to greater presence in the cells interior. The viability and apoptosis assays demonstrated that the mesenchymal stem cells labeled and not labeled respectively with the superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine continue to proliferate over seven days and the percentage of cells in early or late apoptosis is low compared to the percentage of live cells over the three days. CONCLUSION Our results showed that the use of poly-L-lysine complexed with superparamagnetic iron oxide nanoparticles/dextran provides better internalization of these superparamagnetic iron oxide nanoparticles in mesenchymal stem cells Thus, we demonstrated that this type of labeling is not cytotoxic to the mesenchymal stem cells, since the viability and apoptosis assays showed that the cells remain alive and proliferating. The efficiency of this type of labeling in mesenchymal stem cells can provide non-invasive methods for monitoring these cells in vivo.

Acid-sensitive lipidated doxorubicin prodrug entrapped in nanoemulsion impairs lung tumor metastasis in a breast cancer model

AIM To develop an acid-sensitive lipidated, doxorubicin (Dox) prodrug (C16-Dox) to be entrapped in lipid nanoemulsion (NE-C16-Dox) as a nanocarrier to treat breast cancer models (in vitro and in vivo). RESULTS We report the efficacy of NE-C16-Dox in in vitro experiments, as well as the improved chemotherapeutic index and tumor-control efficacy compared with treatment with free Dox in an in vivo murine 4T1 breast cancer model. In addition, NE-C16-Dox allowed the use of a higher dose of Dox, acceptable biocompatibility and a significant reduction in lung metastasis. CONCLUSION Taken together, these results indicate that NE-C16-Dox is promising for breast cancer treatment, thus creating possibilities to translate these nanotechnology concepts to clinical applications.

Mesenchymal stem cell-like properties of CD133+ glioblastoma initiating cells

Glioblastoma is composed of dividing tumor cells, stromal cells and tumor initiating CD133+ cells. Recent reports have discussed the origin of the glioblastoma CD133+ cells and their function in the tumor microenvironment. The present work sought to investigate the multipotent and mesenchymal properties of primary highly purified human CD133+ glioblastoma-initiating cells. To accomplish this aim, we used the following approaches: i) generation of tumor subspheres of CD133+ selected cells from primary cell cultures of glioblastoma; ii) analysis of the expression of pluripotency stem cell markers and mesenchymal stem cell (MSC) markers in the CD133+ glioblastoma-initiating cells; iii) side-by-side ultrastructural characterization of the CD133+ glioblastoma cells, MSC and CD133+ hematopoietic stem cells isolated from human umbilical cord blood (UCB); iv) assessment of adipogenic differentiation of CD133+ glioblastoma cells to test their MSC-like in vitro differentiation ability; and v) use of an orthotopic glioblastoma xenograft model in the absence of immune suppression. We found that the CD133+ glioblastoma cells expressed both the pluripotency stem cell markers (Nanog, Mush-1 and SSEA-3) and MSC markers. In addition, the CD133+ cells were able to differentiate into adipocyte-like cells. Transmission electron microscopy (TEM) demonstrated that the CD133+ glioblastoma-initiating cells had ultrastructural features similar to those of undifferentiated MSCs. In addition, when administered in vivo to non-immunocompromised animals, the CD133+ cells were also able to mimic the phenotype of the original patients tumor. In summary, we showed that the CD133+ glioblastoma cells express molecular signatures of MSCs, neural stem cells and pluripotent stem cells, thus possibly enabling differentiation into both neural and mesodermal cell types.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

Background Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Lipid nanoemulsion passive tumor accumulation dependence on tumor stage and anatomical location: a new mathematical model for in vivo imaging biodistribution studies

Nanoparticle delivery to tumor tissue is one of the most important applications of nanomedicine. However, the literature shows that this pharmacological event is highly-affected by several tumor biology characteristics, including tumor size and maturation. Thus, the objective of the present study is to report on the investigation of the biodistribution of a lipid nanoemulsion (NE) in a breast cancer tumor model using in vivo imaging techniques. As highlights of this study, we can indicate that the biodistribution was measured in different tumor sites (primary and metastatic tumors) and in the same experimental mice for four subsequent weeks. With this approach it is possible to observe that the NE tumor delivery is significantly altered during tumor growth and metastasis progression. Furthermore, in the present report we introduce a phenomenological mathematical model that successfully explains the delivery behavior of a hydrophobic infrared fluorescent NE marker to both primary tumor and metastatic lesions. We believe that these data, in addition to the phenomenological mathematical model, are relevant to understanding how the stage of tumor development can alter macromolecule and/or nanoparticle delivery to tumor tissues, thus improving the efficacy of the passive delivery features promoted by tumor biology.