ABSTRACT
INTRODUCTION: The human bone marrow microenvironment is composed of biological, chemical and physical factors that act in a synergistic way to modulate hematopoietic stem cell biology, such as mesenchymal stromal cells (MSCs), endothelial cells (ECs) and low oxygen levels; however, it is difficult to mimic this human microenvironment in vitro. METHODS: In this work, we developed 3D multicellular spheroid (3D-MS) for the study of human hematopoietic stem cells (HSCs) with some components of perivascular niche. HSCs were isolated from umbilical cord blood, MSCs were isolated from human bone marrow and a microvasculature EC line (CC-2811, Lonza®) was used. For the formation of a 3D structure, a magnetic levitation culture system was used. Cultures were maintained in 21%, 3% and 1% O2 for 15 days. Culture volume, sphericity index and cell viability were determined. Also, human HSC proliferation, phenotype and production of reactive oxygen species were evaluated. RESULTS: After 15 days, 3D-MS exhibited viability greater than 80%. Histology results showed structures without necrotic centers, and higher cellular proliferation with 3% O2. An increase in the expression of the CD34 antigen and other hematopoietic antigens were observed to 1% O2 with MSCs plus ECs and low ROS levels. CONCLUSION: These findings suggest that 3D-MS formed by MSCs, ECs and HSCs exposed to low concentrations of oxygen (1-3% O2) modulate human HSC behavior and mimics some features of the perivascular niche, which could reduce the use of animal models and deepen the relationship between the microenvironment of HSC and human hematological diseases development.
ABSTRACT
Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus.
Subject(s)
Dengue Virus , Endothelial Cells/metabolism , Extracellular Vesicles/virology , Macrophages/ultrastructure , Antigens, CD/metabolism , Cadherins/metabolism , Cell Adhesion Molecules/metabolism , Cytokines/metabolism , Dengue/immunology , Dengue Virus/immunology , Endothelial Cells/immunology , Extracellular Vesicles/physiology , Humans , Macrophages/virology , Permeability , U937 CellsABSTRACT
Introducción. El objetivo de este estudio es analizar los resultados obtenidos con la rectosigmoidectomía perineal con levatorplastia anterior, según la técnica de Altemeier, en un grupo determinado de pacientes de edad avanzada y en los que el riesgo quirúrgico y anestésico era muy elevado. De igual forma, se estudia el porcentaje de enfermedades del suelo pélvico asociadas a estos pacientes. Pacientes y método. En el período comprendido entre 1983 y 2002 se estudió retrospectivamente a 68 pacientes, de los que 45 fueron tratados mediante resección perineal por reunir las condiciones de edad avanzada y alto riesgo quirúrgico. Se analizan los resultados, así como las enfermedades perineales asociadas. Se estudian las complicaciones inmediatas y tardías, y la morbimortalidad en dicho grupo de pacientes. Resultados. Durante un seguimiento promedio de 6 años no se ha registrado mortalidad en el grupo total de pacientes y la morbilidad ha sido escasa y de poca relevancia clínica. Sólo se ha observado recidiva en 2 casos (4,4 por ciento) y el tratamiento realizado fue una nueva resección perineal. La frecuencia de enfermedades del suelo pélvico asociadas ha sido del 88,8 por ciento. Conclusiones. El prolapso de recto se asocia frecuentemente con enfermedades multiorgánicas del suelo pélvico. La resección perineal de Altemeier ofrece buenos resultados y permite intervenir a este grupo de pacientes con una mortalidad nula y una morbilidad poco relevante, así como también tratar otras enfermedades del suelo pélvico asociadas (AU)
