A Rat Treated with Mesenchymal Stem Cells Lives to 44 Months of Age

AbstractThere is a growing interest in the potential of mesenchymal stem cells (MSCs) for implementing regenerative medicine. We assessed the effect of intravenous administration of human bone marrow-derived MSC on the life span of a single Sprague-Dawley female rat. The treatment was started when the rat was 6 months old and the cells were administered every 2 weeks afterward. The treatment did not induce any obvious changes in body growth or behavior and the rat showed the typical age changes for this strain, except that, unlike intact counterparts, the animal did not develop mammary tumors or pituitary gland hyperplasia. The more remarkable effect of the treatment was on life span, which was 44 months compared with an average of 36 months for intact laboratory rats. We conclude that despite the low N value, it is likely that the MSC treatment was responsible for the exceptionally long survival of the rat. The potential rewards of confirming the present findings warrant further studies involving higher N values.Fil: Mansilla, Eduardo. Provincia de Buenos Aires. Ministerio de Salud. Centro Único Coordinador de Ablación e Implante Provincia de Buenos Aires; ArgentinaFil: Roque, Gustavo. Provincia de Buenos Aires. Ministerio de Salud. Centro Único Coordinador de Ablación e Implante Provincia de Buenos Aires; ArgentinaFil: Sosa, Yolanda Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Tarditti, Adrian. Provincia de Buenos Aires. Ministerio de Salud. Centro Único Coordinador de Ablación e Implante Provincia de Buenos Aires; ArgentinaFil: Goya, Rodolfo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentin

A Rat Treated with Mesenchymal Stem Cells Lives to 44 Months of Age

There is a growing interest in the potential of mesenchymal stem cells (MSCs) for implementing regenerative medicine. We assessed the effect of intravenous administration of human bone marrow-derived MSC on the life span of a single Sprague-Dawley female rat. The treatment was started when the rat was 6 months old and the cells were administered every 2 weeks afterward. The treatment did not induce any obvious changes in body growth or behavior and the rat showed the typical age changes for this strain, except that, unlike intact counterparts, the animal did not develop mammary tumors or pituitary gland hyperplasia. The more remarkable effect of the treatment was on life span, which was 44 months compared with an average of 36 months for intact laboratory rats. We conclude that despite the low N value, it is likely that the MSC treatment was responsible for the exceptionally long survival of the rat. The potential rewards of confirming the present findings warrant further studies involving higher N values.Instituto de Investigaciones Bioquímicas de La Plat

Transferência gênica assistida por campos magnéticos: Estudos em células gliais

Las nanopartículas magnéticas (MNP) complejadas con vectores génicos pueden, en presencia de un campo magnético externo, amplificar sustancialmente la eficiencia de la transferencia génica. Esta técnica, denominada magnetofección, es de gran interés en el campo de la terapia génica. En este estudio se caracterizó la mejora de transferencia génica en células gliales B92 utilizando complejos constituidos por diferentes proporciones de MNP asociadas a dos vectores adenovirales, a saber: los complejos entre las MNP denominadas PEI-Mag2 asociadas al adenovector RAd-GFP que expresa la proteína fluorescente verde GFP o al adenovector RAd-DsRed que expresa la proteína fluorescente roja DsRed2. Se demostró que para ambos vectores, a medida que la relación MNP/partícula viral física (PVF) va aumentando, la amplificación de la transfección también aumenta hasta que se llega a una relación MNP/PVF a partir de la cual el factor de amplificación alcanza un plateau. Se determinó que para el complejo PEI-Mag2/RAd-GFP la relación a partir de la cual se alcanza el plateau es de aproximadamente 0,5 fg Fe/PVF mientras que para el complejo PEI-Mag2/RAd-DsRed, esta relación corresponde a aproximadamente 71 fg Fe/PVF. Se concluye que los dos complejos magnéticos estudiados representan promisorias herramientas para mejorar la eficiencia en la terapia génica en células cerebrales.Fil: Pardo, Joaquin. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - la Plata. Instituto de Invest.bioquimicas de la Plata;Fil: Sosa, Yolanda Elena. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - la Plata. Instituto de Invest.bioquimicas de la Plata;Fil: Reggiani, Paula Cecilia. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - la Plata. Instituto de Invest.bioquimicas de la Plata;Fil: Arciniegas, M. I.. Universidad Nacional de la Plata. Facultad de Ciencias Exactas; Argentina;Fil: Sanchez, Francisco Homero. Universidad Nacional de la Plata. Facultad de Ciencias Exactas; Argentina;Fil: Goya, Rodolfo Gustavo. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - la Plata. Instituto de Invest.bioquimicas de la Plata

Stress-induced Gene Expression Sensing Intracellular Heating Triggered by Magnetic Hyperthermia

It is known that alternating magnetic field applications on eukaryotic cells loaded with single domain iron oxide nanoparticles result in high hyperthermic citotoxicity leading to cell dead. Although magnetic hyperthermia therapy for cancer tumours is being developed under this idea, some in vitro assays have shown controversial results indicating that alternating magnetic field triggers large apoptotic effect without significant culture-temperature increase. In agreement with these observations a huge lowering in nanoparticle specific heating rates, when going from the colloidal suspension to cell endosomes, together with cell death, has been reported. Here, we propose a new methodology to determine the occurrence of local heating in cells when alternating magnetic fields in the radiofrequency field range are applied to cell cultures holding very low iron oxide concentrations, being these concentrations insufficient to produce a global cell-culture temperature increase up to therapeutic values. To this end, human lung adenocarcinoma cells (A549 cell line) were transduced with a lentiviral vector encoding the expression of the enhanced green fluorescence protein, EGFP, under the action of the inducible human heat shock protein 70B promoter. This modified A549 cell line was incubated with aqueous suspensions of magnetite core nanoparticles (uncoated or covered with coating agents like citric acid or silicon oxide), and exposed to radiofrequency fields. The application of an alternating magnetic field to cell cultures loaded with nanoparticles resulted in no global temperature increase but EGFP expression. Stress-inducible gene expression scales with uptake and nanoparticle properties like saturation magnetization and heat dissipation efficiency. Our analysis demonstrates that EGFP expression is linked to a localized intracellular temperature increase.Fil: de Sousa, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; ArgentinaFil: Carrea, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Mendoza Zélis, Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; ArgentinaFil: Muraca, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidade Estadual de Campinas; BrasilFil: Mykhaylyk, Olga. Technische Universitat Munchen; AlemaniaFil: Sosa, Yolanda Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Goya, Rodolfo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Sánchez, Francisco Homero. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; ArgentinaFil: Dewey, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Fernández van Raap, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física La Plata. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física La Plata; Argentin

Studies on the gonadotropin-releasing activity of thymulin: Changes with age

We assessed the ability of thymulin, a zinc-dependent nonapeptide produced by the thymic epithelial cells, to influence the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from dispersed anterior pituitary (AP) cells from young, adult, and senescent female rats. Perifusion of young and senescent AP cells with thymulin doses of 10-6 to 10-5 M gave a significant stimulatory response for LH but not FSH. Gonadotropin release was always lower in the senescent cells. AP cells from both age groups incubated with 10-8 to 10-3 M thymulin showed a time- and dose-dependent response for both gonadotropins, with a maximal stimulation at 10-7 M. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with the secretagogue gonadotropin-releasing hormone (GnRH) revealed a synergistic effect on LH release and an additive effect on the release of FSH. The calcium chelator EGTA blocked the gonadotropin-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF, and forskolin significantly increased the thymulin-stimulated release of gonadotropins. The inositol phosphate enhancer LiCl potentiated the action of thymulin on gonadotropins. It is concluded that the gonadotropin-releasing activity documented here for thymulin is an age- and receptor-dependent effect mediated in part by calcium, cAMP, and inositol phosphates.Instituto de Investigaciones Bioquímicas de La Plat

Transferência gênica assistida por campos magnéticos: estudos em células gliais

Las nanopartículas magnéticas (MNP) complejadas con vectores génicos pueden, en presencia de un campo magnético externo, amplificar sustancialmente la eficiencia de la transferencia génica. Esta técnica, denominada magnetofección, es de gran interés en el campo de la terapia génica. En este estudio se caracterizó la mejora de transferencia génica en células gliales B92 utilizando complejos constituidos por diferentes proporciones de MNP asociadas a dos vectores adenovirales, a saber: los complejos entre las MNP denominadas PEI-Mag2 asociadas al adenovector RAd-GFP que expresa la proteína fluorescente verde GFP o al adenovector RAd-DsRed que expresa la proteína fluorescente roja DsRed2. Se demostró que para ambos vectores, a medida que la relación MNP/partícula viral física (PVF) va aumentando, la amplificación de la transfección también aumenta hasta que se llega a una relación MNP/PVF a partir de la cual el factor de amplificación alcanza un plateau. Se determinó que para el complejo PEI-Mag2/RAd-GFP la relación a partir de la cual se alcanza el plateau es de aproximadamente 0,5 fg Fe/PVF mientras que para el complejo PEI-Mag2/RAd-DsRed, esta relación corresponde a aproximadamente 71 fg Fe/PVF. Se concluye que los dos complejos magnéticos estudiados representan promisorias herramientas para mejorar la eficiencia en la terapia génica en células cerebrales.It is known that certain types of magnetic nanoparticles (MNPs) complexed to gene vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells. This technique, called magnetofection, is of great relevance to gene therapy. In the present study the ability of MNP/adenovector complexes to enhance gene transfer to B92 glial cells was assessed. Two complexes were assessed, namely PEI-Mag2/RAd-GFP and PEI-Mag2/RAd-DsRed, which are constituted by the MNP PEI-Mag2 complexed to the adenovector RAd-GFP (expressing the green fluorescent protein GFP) and RAdDsRed (expressing the red fluorescent protein DsRed2), respectively. It was shown that for both vectors, an increase in the ratio MNP/PVP (physical viral particle) is paralleled by an increase in transduction efficiency, up to a certain threshold value at which an efficiency plateau is reached. This threshold value was 0.5 fg Fe/PVP for the RAd-GFP complex and about 71 fg Fe/PVP for the RAd-DsRed complex. It can be concluded that both magnetic complexes assessed in this study represent promising tools for enhancing the efficiency of gene therapy in brain cells.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Física La Plat

Stress-induced Gene Expression Sensing Intracellular Heating Triggered by Magnetic Hyperthermia

It is known that alternating magnetic field applications on eukaryotic cells loaded with single domain iron oxide nanoparticles result in high hyperthermic citotoxicity leading to cell dead. Although magnetic hyperthermia therapy for cancer tumours is being developed under this idea, some in vitro assays have shown controversial results indicating that alternating magnetic field triggers large apoptotic effect without significant culture-temperature increase. In agreement with these observations a huge lowering in nanoparticle specific heating rates, when going from the colloidal suspension to cell endosomes, together with cell death, has been reported. Here, we propose a new methodology to determine the occurrence of local heating in cells when alternating magnetic fields in the radiofrequency field range are applied to cell cultures holding very low iron oxide concentrations, being these concentrations insufficient to produce a global cell-culture temperature increase up to therapeutic values. To this end, human lung adenocarcinoma cells (A549 cell line) were transduced with a lentiviral vector encoding the expression of the enhanced green fluorescence protein, EGFP, under the action of the inducible human heat shock protein 70B promoter. This modified A549 cell line was incubated with aqueous suspensions of magnetite core nanoparticles (uncoated or covered with coating agents like citric acid or silicon oxide), and exposed to radiofrequency fields. The application of an alternating magnetic field to cell cultures loaded with nanoparticles resulted in no global temperature increase but EGFP expression. Stress-inducible gene expression scales with uptake and nanoparticle properties like saturation magnetization and heat dissipation efficiency. Our analysis demonstrates that EGFP expression is linked to a localized intracellular temperature increase.Facultad de Ciencias ExactasInstituto de Física La PlataFacultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitarygonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5′- ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3′) encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymuspituitary-gonadal axis.Instituto de Investigaciones Bioquímicas de La Plat

Gene Therapy in the Neuroendocrine System

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fak/fak) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.Instituto de Investigaciones Bioquímicas de La Plat