Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo: a preliminary report

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a > 50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.

Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo: a preliminary report

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a > 50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.

Decreased plasma ADAMTS-13 activity as a predictor of postoperative bleeding in cyanotic congenital heart disease

OBJECTIVE: To analyze the preoperative plasma antigenic concentration and activity of von Willebrand factor and its main cleaving protease ADAMTS-13 in pediatric patients with cyanotic congenital heart disease undergoing surgical treatment and investigate possible correlations with postoperative bleeding. METHODS: Plasma antigenic concentrations (von Willebrand factor:Ag and ADAMTS-13:Ag) were measured using enzyme-linked immunoassays. Collagen-binding assays were developed to measure biological activities (von Willebrand factor:collagen binding and ADAMTS-13 activity). The multimeric structure of von Willebrand factor was analyzed using Western immunoblotting. Demographic, diagnostic, and general and specific laboratory data and surgery-related variables were subjected to univariate, bivariate, and multivariate analysis for the prediction of postoperative bleeding. RESULTS: Forty-eight patients were enrolled, with ages ranging from 9 months to 7.6 years (median 2.5 years). The plasma concentrations of von Willebrand factor:Ag and ADAMTS-13:Ag were decreased by 65 and 82%, respectively, in the patients compared with the controls (p<0.001). An increased density of low-molecular-weight fractions of von Willebrand factor, which are suggestive of proteolytic degradation (p = 0.0081), was associated with decreased ADAMTS-13 activity, which was likely due to ADAMTS-13 consumption (71% of controls, p = 0.0029) and decreased von Willebrand factor:collagen binding (76% of controls, p = 0.0004). Significant postoperative bleeding occurred in 13 patients. The preoperative ADAMTS-13 activity of <64.6% (mean level for the group), preoperative activated partial thromboplastin time, and the need for cardiopulmonary bypass were characterized as independent risk factors for postoperative bleeding, with respective hazard ratios of 22.35 (95% CI 1.69 to 294.79), 1.096 (95% CI 1.016 to 1.183), and 37.43 (95% ...

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorpórate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Características biológicas das células-tronco mesenquimais: [revisão] / Biological characteristics of mesenchymal stem cells: [review]

Células-tronco são células indiferenciadas. Como tal, apresentam uma série de características que as tornam candidatas à utilização terapêutica. As principais características das células-tronco são a capacidade de autorrenovação e de se diferenciarem em diversos tipos celulares. Desta forma, acredita-se que células-tronco presentes nos diferentes tecidos tenham papel regenerativo quando estes sofrem uma lesão ou injúria. Entre os tecidos conhecidos por apresentarem células-tronco após a vida pós-natal, a medula óssea foi a mais estudada, por muitos anos, como fonte tanto de células-tronco hematopoéticas quanto de células-tronco mesenquimais, também denominadas de células mesenquimais estromais da medula óssea ou células estromais mesenquimais multipotentes. Estas células são um grupo de células clonogênicas, presentes no estroma da medula óssea, que, quando submetidas a diferentes estímulos apropriados, são capazes de se diferenciarem em várias linhagens de células, como a osteogênica, a condrogênica e a adipogênica e, possivelmente, em outros tipos celulares não mesodérmicos, como células neurais ou hepatócitos. Nesta revisão, as principais características das células-tronco mesenquimais serão abordadas, incluindo os marcadores moleculares e de membrana, as características de divisão e de diferenciação, a heterogeneidade e as aplicações clínicas potenciais.

Stem cells are undifferentiated cells. They show various characteristics that make them suitable for clinical applications. The main stem cell characteristics are their capacity of autorenewal and of differentiation into different cell lines so it is quite possible that stem cells in different tissues exhibit a regenerative role when these tissues are injured. Bone marrow is the best studied tissue as a source of hematopoietic stem cells as well as mesenchymal stem cells (also known as mesenchymal stromal cells or mesenchymal stromal multipotent cells); clonogenic cells in the bone marrow stroma. They are able to differentiate under specific stimuli in several cell lines including osteogenic, chondrogenic and adipogenic cells, and probably in other nonmesodermic cell lines such as neural cells or hepatocytes. Here the main characteristics of mesenchymal stem cells will be discussed, including the molecular and membrane markers, the division and differentiation properties, the heterogeneity, and the potential clinical applications.

Células-tronco do líquido amniótico / Amniotic fluid stem cells

Desde o primeiro isolamento e cultivo de células-tronco embrionárias humanas, há mais de 10 anos, seu uso na pesquisa e terapia foi inibida por considerações éticas complexas e pelo risco de transformação maligna destas células indiferenciadas após transplante no paciente. As células-tronco adultas são eticamente aceitas e o risco de transformação maligna é muito baixo. Entretanto, seu potencial de diferenciação e sua capacidade proliferativa são limitados. Cerca de 6 anos atrás, a descoberta de célulastronco no líquido amniótico que expressavam Oct-4, um marcador específico de pluripotencialidade, com alta capacidade de proliferação e diferenciação, iniciou um novo campo promissor na área das células-tronco. Estas células têm potencial de se diferenciar em células dos três folhetos germinativos. Não formam tumores in vivo e não levantam os questionamentos éticos associados com as células-tronco embrionárias humanas. Futuras investigações revelarão se as células-tronco do líquido amniótico realmente irão representar um tipo intermediário com vantagens em relação tanto às células-tronco embrionárias quanto às adultas. Este artigo faz uma revisão acerca destes tópicos e das características biológicas das células-tronco do líquido amniótico.

Since the first successful isolation and cultivation of human embryonic stem cells about 10 years ago, their use for research and therapy has been constrained by complex ethical considerations as well as by the risk of development of malignancies of undifferentiated embryonic stem cells after transplantation into the patient. Adult stem cells are ethically acceptable and the risk of tumor development is low. However, their differentiation potential and proliferative capacity are limited. About 6 years ago, the discovery of Oct-4 expressing amniotic fluid stem cells, a specific marker of pluripotency, with a high proliferative capacity, and multilineage differentiation potential, initiated a promising field of research. These cells, indeed, have the potential to differentiate into cells of all three embryonic germ layers. They do not form tumors in vivo and do not raise ethical concerns. Further investigation will reveal whether these cells really are an intermediate cell type with advantages over both embryonic and adult stem cells. This article reviews the biological characteristics of amniotic fluid stem cells.

Pfaffia paniculata extract improves hydratation of eritrocytes in vitro and sickle cell disease symptoms

Purpose: Pfaffia paniculata, a plant known as Brazilian ginseng, has been claimed by some patients suffering from sickle cell disease to have beneficial effects. In order to examine this assertion, a powder extract was obtained from the roots of the plant. Pacients and methods. The extract was studied to verified the desickling properties in vitro. We studied the behavior of blood cells treated with Pfaffia paniculate extract in vitro through morpholofic analyses of blood cells incubated with paffia paniculate extract in vitro. Thirty Brazilian patients with sickle cell disease receiving capsules containing the powder extract of Pfaffia paniculata (500 mg each) every 8 hours or capsules containing placebo were followed up for three months. The number of erythrocytes, reticulocytes, sickle cells, and peripheral erythroblast, hemoglobin (HB), hematocrit (HT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), were determined in peripheral blood immediately before and 2 and 3 months after the beginning of the treatment. Results: Administration of Brazilian ginseng powder extract to patients having sickle cell resulted in decrease in sickle cells, erythroblast and reticulocytes in blood as well as increase in hemoglobin and hematocrit levels. Conclusion: The Pfaffic extract exhibited desickling properties when incubated with blood cells from deasese sickle cell disease patients or blood cells treated with 2% sodium methabisulphite. The clinical findings showed that treatment also led to improvement of the sintoms and signs in these patients.

Resistência à proteína C ativada / Resistance to activeted protein C

O fator V da coagulaçäo, quando ativado, participa como um co-fator essencial na ativaçäo da pró-trombina pelo fator X ativado. O fator V ativado é inativado pela proteína C ativada, numa reaçäo potencializada pela proteína S. Uma mutaçäo no éxon 10 (Arg 506 Gln) do gene do fator V dß origem a uma molécula do fator V que näo é adequadamente inativada pela proteína C ativada, o que causa a chamada resistência à proteína C ativada.

Efectos adversos en la recuperación hematológica después del trasplante de médula ósea alogénica / Adverse effects in hematological recovery following an allogenic bone marrow transplantation

La recuperación hematológica es un elemento decisivo para el éxito del trasplante de médula ósea. Algunos factores pueden afectar este período de recuperación lo que prolonga el tiempo necesario para la implantación de la médula. Estos factores se analizaron en 5 pacientes con anemia aplástica y en 5 con leucemias, en quienes se hicieron 13 trasplantes de médula ósea. En 2 pacientes con leucemia mieloide crónica y fibrosis intensa de la médula ósea se observó un retardo de la implantación del injerto, por lo que recibieron una segunda infusión medular procedente de los mismos donantes iniciales. En un paciente con leucemia mieloide aguda hubo persistencia de isohemaglutininas contra las células del donante, enfermedad del injerto contra huésped crónica y una pobre punción medular tras el trasplante. Los pacientes que no recibieron metotrexate como profilaxis para la enfermedad de injerto contra huésped, tuvieron al parecer una recuperación medular más rápida y hubo una diferencia significativa en cuanto al día postrasplante en que el conteo de granulocitos superó la cifra de 1 x 10 a la 9/L (p < 0,1). La infección por citomegalovirus se asoció con un empeoramiento de la función medular. Algunos de estos factores ocasionaron una prolongación de los requerimientos transfusionales y fueron elementos adversos en la evolución del trasplante