{"id":115759,"date":"2014-06-06T00:00:00","date_gmt":"2014-06-06T00:00:00","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/trasplante-de-celulas-mensequimales-fucosiladas-en-el-tratamiento-de-la-osteoporosis-estudio-precla%c2%adnico-y-prueba-de-concepto\/"},"modified":"2014-06-06T00:00:00","modified_gmt":"2014-06-06T00:00:00","slug":"trasplante-de-celulas-mensequimales-fucosiladas-en-el-tratamiento-de-la-osteoporosis-estudio-precla%c2%adnico-y-prueba-de-concepto","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/osteopatologia\/trasplante-de-celulas-mensequimales-fucosiladas-en-el-tratamiento-de-la-osteoporosis-estudio-precla%c2%adnico-y-prueba-de-concepto\/","title":{"rendered":"Trasplante de c\u00e9lulas mensequimales fucosiladas en el tratamiento de la osteoporosis. estudio precl\u00ednico y prueba de concepto"},"content":{"rendered":"<h2>Tesis doctoral de <strong> Valentin Caba\u00f1as Perianes <\/strong><\/h2>\n<p>Abstract objectives: osteoporosis is a skeletal disease characterized by systemic bone loss with an increased risk to bone fractures, associated with high morbidity and mortality. Mesenchymal stem cells (msc) from bone marrow (bm) are ideal candidates for the treatment of osteoporosis because they are able to differentiate to osteoblasts. Although bone graft of mscs has been demonstrated, its osteotropism is low when they are administered intravenously (iv), of choice in this multifocal disease. Msc homing depends upon the adhesive interaction between msc surface receptors and counter receptors on endothelial cells. E-selectin protein is induced on microvascular endothelium in inflammatory tissues in response to cytokines and it is constitutively expressed in bm endothelial cells. The hematopoietic cell e and l-selectin ligand (hcell), a glycoform of cd44, is a potent e-selectin ligand expressed constitutively in the hematopoietic stem cells (hsc). Hcell represents the key to involve in cell recruitment to the bm. Msc express cd44 but do not express hcell or other e-selectin ligands. Ex vivo fucosylation (adding a fucose residue) in ?1-3 position of the cd44 antigen by use of the enzyme fucosyltransferase vi (ftvi) yields hcell in msc increasing the affinity for e-selectin and osteotropism. The aim of this preclinical study was to describe the safety and efficacy of human fucosylated msc infused in an immunocompromised mice model (nod\/scid).  Methods:  31 nod\/scid mice were randomized to by tail vein injection:  1&#215;106 fucosylated msc (n=13), 1&#215;106 msc (n=14) or saline (n=4). Toxicities were evaluated by a clinical score, weight and histological assessment (heart, lung, liver, spleen, kidney, gonads, brain, bone and bm).  Rt-pcr array-based evaluation of the expression of human ?-Actin and ?2-microglobulin genes was performed to study biodistribution of msc. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype. Additional samples of tibia and calota were to immunostained with a polyclonal rabbit anti-human osteocalcina to demonstrate efficacy. Osteocalcin-positive cells were identified by a dark-brown cytoplasmic precipitate. Results: there was no unexpected death and none of the mices had any acute toxicity. One mouse in the saline arm had mild chronic toxicity primarily manifested as piloerection, and moderate changes in behavior. The histology of heart, liver, kidney, spleen, gonads, brain, bone and bone marrow was normal in all mice. There were localized areas of lung inflammation in 15%, 42% and 25% of mice infused with fucosylated msc, msc, and saline, respectively without significance differences (p=0.28).  Biodistribution was normal with except  one mice (infused with msc non-fucosylated) showed an expression of human rna ?-Actin and ?2-microglobulin in the lung sample taken after 12 weeks of intravenous infusion .Osteoblasts were seen in 100% of mices infused with fucosylated msc, in 62.5%% of animals infused with msc alone, and none in saline group (p=0.01). Nod\/scid mice infused with fucosylated msc presented a higher number of osteoblasts positive for osteocalcin in 10 high-power fields (400x) of  tibia and calvarium sections that non-fucosylated (32 vs 5.5) (p=0.0082). Human osteoblasts were detected inside the bone from the fifth to the twelfth week after infusion. Conclusions: our preclinical trial demonstrate that the intravenous infusion of human fucosylated msc is safe and effective in guiding the cells to bone with a higher potential to ossification in nod\/scid mices that non-fucosylated msc. These results are allowing to start a human clinical trial with intravenous fucosylated msc as treatment in patients with osteoporosis.     resumen:  objetivos: la osteoporosis es una enfermedad \u00f3sea definida por la p\u00e9rdida de masa \u00f3sea generalizada  con un elevado riesgo de fracturas \u00f3seas asociado a una alta morbimortalidad. Las c\u00e9lulas stem mesenquimales (csm) de m\u00e9dula \u00f3sea (mo) pueden representar un tratamiento \u00f3ptimo para la osteoporosis debido a su capacidad innata de diferenciaci\u00f3n a osteoblastos. Aunque el injerto de las csm ha sido demostrado, su tropismo \u00f3seo es bajo cuando se administran de forma intravenosa, imprescindible para el tratamiento de esta enfermedad sist\u00e9mica. Su tropismo depende de la interacci\u00f3n de sus receptores de superficie con los que se encuentran en las c\u00e9lulas endoteliales. La prote\u00edna e-selectina es inducida en el endotelio de la microvasculatura de tejidos inflamatorios en respuesta a citoquinas y es constitutivamente expresada en las c\u00e9lulas endoteliales de mo. La glicoforma de cd44 (hcell), un potente ligando de e y l-selectina, es expresada de forma innata por las c\u00e9lulas stem hematopoy\u00e9ticas (csh). Hcell es la mol\u00e9cula clave para el reclutamiento celular hacia la mo. Las csm expresan cd44 pero no hcell ni otros ligandos de e-selectina de forma constitutiva.  La fucosilaci\u00f3n (adici\u00f3n de una fucosa) ex vivo en posici\u00f3n  ?1,3 del ant\u00edgeno cd44 mediante la enzima fucosiltransferasa vi (ftvi) produce hcell en las csm, incrementando su afinidad por e-selectina y por tanto su osteotropismo. El objetivo de este estudio precl\u00ednico es evaluar la seguridad y la eficacia de las csm humanas fucosiladas en un modelo murino inmunocomprometido (nod\/scid). M\u00e9todos: 31 ratones nod\/scid fueron randomizados a recibir de forma intravenosa: 1&#215;106 csm fucosiladas (n=13), 1&#215;106 csm (n=14), o salino (n=4). Las toxicidades fueron evaluadas por un score cl\u00ednico, peso y un examen histol\u00f3gico (coraz\u00f3n, pulm\u00f3n, h\u00edgado, bazo, ri\u00f1\u00f3n, g\u00f3nadas, cerebro, hueso y mo). Se realiz\u00f3 estudio de biodistribuci\u00f3n mediante detecci\u00f3n de los genes ?-Actina y ?2-microglobulina humanas. La integridad gen\u00e9tica fue evaluada por cariotipo. Se realiz\u00f3 inmunohistoqu\u00edmica con un anticuerpo policlonal  anti-osteocalcina humana para valorar eficacia. Las c\u00e9lulas osteocalcina positivas fueron identificadas por un precipitado citoplasm\u00e1tico de color marr\u00f3n oscuro. Resultados: no se produjo ning\u00fan fallecimiento inesperado y ning\u00fan rat\u00f3n mostr\u00f3 toxicidad aguda. Un rat\u00f3n en el brazo de salino present\u00f3 toxicidad cr\u00f3nica leve (piloerecci\u00f3n y cambios moderados en el comportamiento).  La histolog\u00eda de coraz\u00f3n, h\u00edgado, bazo, ri\u00f1\u00f3n, cerebro, hueso y mo fue normal en todos los ratones. Se objetivaron \u00e1reas localizadas inflamatorias en los pulmones de un 15%, 42% y 25% de los ratones infundidos con csm fucosiladas, csm y salino respectivamente sin diferencias significativas (p=0.28). La biodistribuci\u00f3n fue normal en todos los ratones con la excepci\u00f3n de un rat\u00f3n (infundido con csm sin fucosilar) que mostr\u00f3 expresi\u00f3n de arn humano (?-Actina and ?2-microglobulina) en la muestra pulmonar tomada a las 12 semanas despu\u00e9s de la infusi\u00f3n. Se observaron osteoblastos en el 100% de los ratones infundidos con csm fucosiladas, en 62,5% de los infundidos con csm, y ninguno en el grupo de salino (p=0.01). Los ratones infundidos con csm fucosiladas presentaron un mayor n\u00famero de osteoblastos osteocalcina positivos en 10 campos de gran aumento (400x) de secciones de tibia y calota con respecto a los infundidos con no fucosiladas (32 vs 5.5) (p=0.0082). Las csm fucosiladas. Los osteoblastos humanos fueron detectados en el hueso desde la quinta a la doceava semana post-infusi\u00f3n. Conclusiones: nuestro ensayo precl\u00ednico demuestra que la infusi\u00f3n intravenosa de csm fucosiladas es segura y eficaz en guiar las c\u00e9lulas al hueso con un potencial m\u00e1s elevado de osificaci\u00f3n en ratones nod\/scid que las csm no fucosiladas. Estos resultados van a permitir comenzar con un ensayo cl\u00ednico en humanos con csm fucosiladas.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Trasplante de c\u00e9lulas mensequimales fucosiladas en el tratamiento de la osteoporosis. estudio precl\u00ednico y prueba de concepto<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Trasplante de c\u00e9lulas mensequimales fucosiladas en el tratamiento de la osteoporosis. estudio precl\u00ednico y prueba de concepto <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Valentin Caba\u00f1as Perianes <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Murcia<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 06\/06\/2014<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Ana Mar\u00eda Garcia Hernandez<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: Juan  jose Lahuerta palacios <\/li>\n<li> Majado mart\u00ednez m. juliana (vocal)<\/li>\n<li>fermin Sanchez-guijo martin (vocal)<\/li>\n<li>Luis Francisco Linares ferrando (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Valentin Caba\u00f1as Perianes Abstract objectives: osteoporosis is a skeletal disease characterized by systemic bone loss with an 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