RESUMEN
Gating of positron emission tomography images has been shown to reduce the motion effects, especially when imaging small targets, such as coronary plaques. However, the selection of optimal number of gates for gating remains a challenge. Selecting too high number of gates results in a loss of signal-to-noise ratio, while too low number of gates does remove only part of the motion. Here, we introduce a respiratory-cardiac motion model to determine the optimal number of respiratory and cardiac gates. We evaluate the model using a realistic heart phantom and data from 12 cardiac patients (47-77 years, 64.5 on average). To demonstrate the benefits of our model, we compared it with an existing respiratory model. Based on our study, the optimal number of gates was determined to be five respiratory and four cardiac gates in the phantom and patient studies. In the phantom study, the diameter of the most active hot spot was reduced by 24% in the dual gated images compared to non-gated images. In the patient study, the thickness of myocardium wall was reduced on average by 21%. In conclusion, the motion model can be used for estimating the optimal number of respiratory and cardiac gates for dual gating.
Asunto(s)
Corazón/diagnóstico por imagen , Corazón/fisiología , Tomografía Computarizada por Tomografía de Emisión de Positrones , Anciano , Algoritmos , Enfermedades Cardiovasculares/diagnóstico por imagen , Femenino , Fluorodesoxiglucosa F18 , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Movimiento (Física) , Fantasmas de Imagen , Respiración , Relación Señal-RuidoRESUMEN
Conversion of the nephrogenic mesenchyme into epithelial tubules requires an inductive stimulus from the ureter bud. Here we show with immunofluorescence techniques that the undifferentiated mesenchyme before induction expresses uniformly type I and type III collagens. Induction both in vivo and in vitro leads to a loss of these proteins and to the appearance of basement membrane components including type IV collagen. This change correlates both spatially and temporally with the determination of the mesenchyme and precedes and morphological events. During morphogenesis, type IV collagen concentrates at the borders of the developing tubular structures where, by electron microscopy, a thin, often discontinuous basal lamina was seen to cover the first pretubular cell aggregates. Subsequently, the differentiating tubules were surrounded by a well-developed basal lamina. No loss of the interstitial collagens was seen in the metanephric mesenchyme when brought into contact with noninducing tissues or when cultured alone. Similar observations were made with nonnephrogenic mesenchyme (salivary, lung) when exposed to various heterotypic tissues known to induce tubules in the nephrogenic mesenchyme. The sequential shift in the composition of the extracellular matrix from an interstitial, mesenchymal type to a differentiated, epithelial type is so far the first detectable response of the nephrogenic mesenchyme to the tubule-inducing signal.
Asunto(s)
Colágeno/metabolismo , Riñón/embriología , Procolágeno/metabolismo , Animales , Inducción Embrionaria , Espacio Extracelular/metabolismo , Riñón/citología , RatonesRESUMEN
The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell-specific small GTPase. Northern blot analysis on various mouse organs, revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport.
Asunto(s)
Polaridad Celular/genética , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP rab , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico/genética , ADN/análisis , Epitelio/química , Biblioteca de Genes , Inmunohistoquímica , Intestinos/química , Riñón/química , Riñón/ultraestructura , Hígado/química , Ratones , Datos de Secuencia MolecularRESUMEN
Local brain tumor therapy using lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2) has not proved to be effective in preliminary clinical trials. One obstacle to effective use of this therapy is ignorance about the events that follow contact of the LAK cells with glioma tissue. We used multicellular spheroids grown from human glioma cell lines as targets to study, in vitro, the effect of LAK cells against three-dimensional glioma tissue. Here we describe the ultrastructural changes in spheroids of H-2 glioma cells incubated in pellets of LAK cells for up to 24 hours. In H-2 spheroids, cellular damage was not restricted to the effector cell-target cell (effector-target) contact; it extended farther, at least partly because of nonspecific changes in the spheroid micromilieu. Formation of cytoplasmic blebs, a characteristic effect of T cells, natural killer cells, and LAK cells on single target cells, also occurs in H-2 spheroids, and it is not limited to the effector-target contact area either. These findings suggest that LAK cells release membrane-damaging agents that remain active outside the effector-target area, in the micromilieu of H-2 spheroid tissue.
Asunto(s)
Glioma/inmunología , Células Asesinas Activadas por Linfocinas/inmunología , Citotoxicidad Inmunológica , Relación Dosis-Respuesta Inmunológica , Glioma/ultraestructura , Humanos , Inmunidad Celular , Técnicas In Vitro , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Células Tumorales CultivadasRESUMEN
Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.
Asunto(s)
Diferenciación Celular/genética , Proteínas del Huevo/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/biosíntesis , Péptidos y Proteínas de Señalización Intercelular , Oocitos/metabolismo , Oogénesis/genética , Folículo Ovárico/citología , Isoformas de Proteínas/biosíntesis , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 15 , Proteínas del Huevo/genética , Etiquetas de Secuencia Expresada , Femenino , Factor 9 de Diferenciación de Crecimiento , Sustancias de Crecimiento/genética , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Oocitos/citología , Folículo Ovárico/metabolismo , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Embryonal carcinoma (EC) cells, which are the malignant stem cells of teratocarcinomas, are considered similar to early embryo cells. The EC cells can be grown in vitro, and many of them can be experimentally induced to differentiate; upon differentiation, the cells become benign. Here we review some of the changes that take place in the cellular and molecular characteristics of murine F9 EC cells as they differentiate into endodermal cells. Upon differentiation of F9 cells, distinct changes occur in their cell surface molecules, cytoskeleton-associated proteins and cell adhesion properties. Simultaneously, the rate of cell proliferation decreases due to a dramatic increase in duration of G1 and S phases of the cell cycle. The changes in gene expression and cell behavior occurring during endodermal differentiation of EC cells closely resemble those occurring when the endoderm differentiates in the embryo. Teratocarcinoma stem cell lines may thus be exploited to enhance understanding of both teratoma-type neoplasms and embryonic development.
Asunto(s)
Diferenciación Celular , Modelos Biológicos , Teratoma/patología , Animales , Adhesión Celular , Proteínas del Citoesqueleto/fisiología , Células Madre de Carcinoma Embrionario , Ratones , Células Madre NeoplásicasRESUMEN
Vesicular transport processes play crucial roles in the biogenesis of cellular membranes and in the polarized transport functions of epithelial cells. During the 1990's we have witnessed major progress in elucidation of the machineries responsible for the intracellular membrane trafficking. The components of these machineries are abundant in tissues with a high content of epithelial cells, such as the kidney. However, the developmental role of the membrane trafficking apparatus in higher eukaryotes has been addressed hardly at all. We summarize here data on the presence and the functional role of vesicle transport proteins in the kidney, and describe work addressing the developmentally regulated expression and localization of three molecules suggested to be involved in polarized trafficking in kidney epithelia, Rab17, syntaxin 3, and Munc-18-2. The results show that specialized transport machinery is induced during differentiation of renal epithelia. However, the expression levels of the components under study are highest in the mature structures, indicating that the proteins are predominantly required for the function of mature epithelia and possibly for the maintenance of the polarized phenotype of specific epithelial cells. The proteins are, however, detected at low levels already in earlier, differentiating structures, and could thus also be involved in the differentiation of kidney epithelia.
Asunto(s)
Vesículas Cubiertas/fisiología , Riñón/embriología , Proteínas del Tejido Nervioso , Proteínas de Transporte Vesicular , Animales , Transporte Biológico Activo/fisiología , Membrana Celular/metabolismo , Vesículas Cubiertas/metabolismo , Inducción Embrionaria , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Munc18 , Proteínas/metabolismo , Proteínas Qa-SNARE , Proteínas de Unión al GTP rab/metabolismoRESUMEN
We used the differential hybridization technique for isolating developmentally regulated genes from the mouse metanephric kidney. In this screening, we identified the cDNA encoding high-mobility-group protein 17 (HMG-17), a chromosomal non-histone protein which modulates the conformation of transcriptionally active chromatin. Using Northern blot analysis, the HMG-17 mRNA was strongly expressed during embryogenesis and downregulated in various adult murine organs. At the histological level, the transcript localized to differentiating tissue regions and was apparently downregulated in mature structures indicating that HMG-17 expression is linked to cell differentiation. HMG-17 can thus be regarded as a general marker for tissues or cells undergoing differentiation during organogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Grupo de Alta Movilidad/genética , Riñón/embriología , Animales , Diferenciación Celular , División Celular , ADN Complementario/genética , Femenino , Marcadores Genéticos , Hibridación in Situ/métodos , Masculino , Mesodermo , Ratones , Ratones Endogámicos CBA , Morfogénesis , Especificidad de Órganos , ARN Mensajero/análisisRESUMEN
High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN.
Asunto(s)
Podocitos/citología , Podocitos/metabolismo , Receptores Toll-Like/antagonistas & inhibidores , Acetatos/farmacología , Animales , Apoptosis/fisiología , Diabetes Mellitus Tipo 1/sangre , Humanos , Lipopolisacáridos/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Oxazoles/farmacología , Podocitos/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Ratas , Transducción de Señal , Receptores Toll-Like/metabolismoRESUMEN
We studied the effect of microtubule-disrupting drugs and cold treatment on the organization of cytoplasmic microtubules and intermediate filaments by the indirect immunofluorescence (IFL) technique. Treatment of the cultured fibroblasts with demecolcine brought about a rapid and complete disruption of microtubules and a concomitant redistribution of intermediate filaments into coiling perinuclear bundles. Cold-treatment of the cells resulted also in a complete disappearance of microtubules but did not cause any change in the distribution of intermediate filaments. During the reorganization process of the cytoplasmic microtubular complex, apparently normal arrays of intermediate filaments were discernible in the cells. The results show that microtubules can undergo a disruption-reorganization process without changes in the organization of intermediate filaments and suggest that the maintenance of normal intermediate filament organization is independent of microtubules.
Asunto(s)
Citoesqueleto/ultraestructura , Microtúbulos/ultraestructura , Células Cultivadas , Frío , Citoesqueleto/efectos de los fármacos , Demecolcina/farmacología , Fibroblastos , Humanos , Microtúbulos/efectos de los fármacosRESUMEN
Recombinant AAV efficacy has been demonstrated in numerous gene therapy preclinical studies. As this vector is increasingly applied to human clinical trials, it is a priority to evaluate the risks of its use for workers involved in research and clinical trials as well as for the patients and their descendants. At high multiplicity of infection, wild-type AAV integrates into human chromosome 19 in approximately 60% of latently infected cell lines. However, it has been recently demonstrated that only approximately 1 out of 1000 infectious units can integrate. The mechanism of this site-specific integration involves AAV Rep proteins which are absent in vectors. Accordingly, recombinant AAV (rAAV) do not integrate site-specifically. Random integration of vector sequences has been demonstrated in established cell lines but only in some cases and at low frequency in primary cultures and in vivo. In contrast, episomal concatemers predominate.Therefore, the risks of insertional mutagenesis and activation of oncogenes are considered low. Biodistribution studies in non-human primates after intramuscular, intrabronchial, hepatic artery and subretinal administration showed low and transient levels of vector DNA in body fluids and distal organs. Analysis of patients body fluids revealed rAAV sequences in urine, saliva and serum at short-term. Transient shedding into the semen has been observed after delivery to the hepatic artery. However, motile germ cells seemed refractory to rAAV infection even when directly exposed to the viral particles, suggesting that the risk of insertion of new genetic material into the germ line is absent or extremely low. Risks related to viral capsid-induced inflammation also seem to be absent since immune response is restricted to generation of antibodies. In contrast, transgene products can elicit both cellular and humoral immune responses, depending on the nature of the expressed protein and of the route of vector administration. Finally, a correlation between early abortion as well as male infertility and the presence of wt AAV DNA in the genital tract has been suggested. Although no causal relationship has been established, this issue stresses the importance of using rAAV stocks devoid of contaminating replication-competent AAV. This review comprehensively examines virus integration, biodistribution, immune interactions, and other safety concerns regarding the wild-type AAV and recombinant AAV vectors.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/efectos adversos , Animales , Formación de Anticuerpos , Dependovirus/fisiología , Inmunidad Celular , Recombinación Genética , Medición de Riesgo , Transgenes , Latencia del VirusRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease characterised by a progressive loss of the dopaminergic neurones in the substantia nigra pars compacta. Accumulating evidence indicates that apoptosis contributes to neuronal cell death in PD patients' brain. Excitotoxicity, oxidative stress, and mitochondrial respiratory failure are thought to be the key inducers of the apoptotic cascade. Even though the initial cause and the mechanism of degeneration are poorly understood, neuroprotection can be achieved by interfering with neuronal cell death either directly or by preventing neuronal dysfunction. Potential agents for neuroprotection are neurotrophic factors, inhibitors of apoptosis or anti-oxidative agents. However, the existence of the blood-brain barrier precludes systemic delivery of these factors. In situ gene delivery provides strategies for local and sustained administration of protective factors at physiologically relevant doses. Viral vectors mediating stable gene expression in the central nervous system exist and are still under development. Efficacy of these vectors has repeatedly been demonstrated in the animal models both ex vivo and in vivo. Ex vivo gene delivery could furthermore be combined with cell replacement therapies by transplanting genetically modified cells compensating for the lost neuronal cell population in order to provide neuroprotection to both the grafted cells and degenerating host neurones. However, several aspects of gene transfer, such as uncontrolled diffusion, axonal transport, unpredictable site of integration and immunological responses, still raise safety concerns and justify further development of viral and non-viral vectors as well as genetic elements with tightly controlled gene expression. Various relevant animal models for Parkinson's disease are available for the evaluation of gene therapy strategies. These include induction of cell death in specific neurone population through administration of toxins either directly in the brain or systemically, as well as transgenic mice expressing human disease-associated mutations.
Asunto(s)
Terapia Genética , Enfermedad de Parkinson/terapia , Animales , Muerte Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Enfermedad de Parkinson/patologíaRESUMEN
We followed the early post-induction changes in nucleic acid synthesis of the metanephric kidney anlage in vitro. Enhanced incorporation of [3H]-thymidine and [3H]-adenine was detected, but several factors were shown to influence the interpretation of such in vitro experiments. The incorporation is dependent not only on the stage of development of the target organ but also on its transfer to organ culture, as early rudiments require an "adaptive" pre-cultivation to stabilize their metabolism; at more advanced stages growth and DNA synthesis proceed without delay. Another potential artifact is radiation damage which is caused by the incorporated radioisotope and can be detected in prolonged cultures. A [3H]-adenine pulse of more than 1 microCi/ml for 2 hr leads to definite growth retardation, and a 10-microCi/ml pulse causes extensive cell death and atrophy on a 4- to 6-day subculture. The radiation damage is dose-dependent and of variable severity in the different cell lineages within the organ. Since the radioisotope doses were in the range of those commonly used for monitoring cell proliferation and metabolism, we stress the risk of obtaining artifactual results, especially in prolonged cultures after pulse-labeling.
Asunto(s)
Adenina/metabolismo , Riñón/metabolismo , Nucleótidos de Adenina/metabolismo , Animales , Diferenciación Celular/efectos de la radiación , División Celular/efectos de la radiación , Núcleo Celular/efectos de la radiación , Núcleo Celular/ultraestructura , ADN/biosíntesis , Riñón/citología , Riñón/embriología , Riñón/efectos de la radiación , Ratones , Microscopía Electrónica , Técnicas de Cultivo de Órganos , ARN/biosíntesis , TritioRESUMEN
Dendritic cells (DC) from human peripheral blood were enriched using a method including adherence on plastic, depletion of phagocytic cells, flotation on density gradient column and panning on antibody-coated surfaces. The course of cell separation was evaluated by characterizing the morphological, antigenic and functional features of the different cell fractions. Using the population depleted of strongly adherent cells as the source, we were able to achieve a cell fraction containing 40-65% of DC (the main contaminants being monocytes and natural killer cells). Functionally these cells were highly stimulatory in autologous mixed leukocyte reaction. On the other hand, cells which primarily adhered well and were detached after overnight culture contained less than 5% of DC (the main contaminants being monocytes) after the same purification protocol. The calculated yields of DC were 1-2 X 10(6) and less than 0.5 X 10(6) from the nonadherent and adherent populations, respectively. Thus we concluded that the adhesiveness of DC from human blood is so weak that they can more efficiently and in a more reproducible way be enriched from the primarily nonadherent cell fraction.
Asunto(s)
Células Dendríticas/citología , Anticuerpos Monoclonales , Adhesión Celular , Separación Celular , Citometría de Flujo , Humanos , Microscopía ElectrónicaRESUMEN
Although targeted gene disruption of GDF-9, an oocyte derived growth factor, leads to an arrest of folliculogenesis and causes infertility in female mice, little is known on the expression of GDF-9 protein in the ovary. We show that GDF-9 protein is expressed in rat oocytes during folliculogenesis from the early primary follicle stage onwards but the most intensive immunostaining was seen in primary and preantral follicles. Northern blot analyses of the ontogeny of GDF-9 gene expression in postnatal rat ovaries showed that the GDF-9 transcript levels are clearly increased on the second postnatal day concomitant with the appearance of primary follicles. Interestingly, Northern blot and in situ hybridization analyses indicate a similar expression pattern for GDF-9B, the rat ortholog of a mouse GDF-9 like factor for which we recently reported the partial amino acid sequence. The polypeptide sequences deduced from isolated ovarian cDNAs indicate that the rat GDF-9 prepropeptide is 440 amino acids (aa) in length and the putative mature peptide is 135 aa whereas rat GDF-9B is 391 aa long and the mature region is 125 aa. We conclude that (1) the GDF-9 protein is highly expressed in the oocytes of primary follicles of rat ovaries suggesting that it plays a role mainly in early folliculogenesis and that (2) the full-length polypeptide sequence of GDF-9B suggests that this novel TGF-beta family member is likely to be a secreted growth factor that may regulate folliculogenesis at similar developmental stages as GDF-9.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Ovario/metabolismo , ARN Mensajero/genética , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 15 , Clonación Molecular , ADN Complementario , Femenino , Factor 9 de Diferenciación de Crecimiento , Sustancias de Crecimiento/química , Ratones , Datos de Secuencia Molecular , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Señales de Clasificación de Proteína/genética , ARN Mensajero/análisis , Ratas , Transcripción Genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Amniotic fluid at 15-20 weeks contains high concentrations of microvillar enzymes which serve as markers for the prenatal diagnosis of cystic fibrosis. We found that these enzymes were mostly bound to amniotic fluid particulate fraction which eluted in the void volume on Sepharose 6B chromatography and precipitated in ultracentrifugation at 100,000g. Electron microscopy showed that the particulate fraction comprised membrane fragments and small vesicles. It represented less than 1% of the total protein in cell free amniotic fluid. Sorbitol gradient centrifugation showed heterogeneity in the particulate fraction. The enzyme patterns of amniotic fluid and meconium particulate fractions resembled each other. Antiserum against particulate fraction in amniotic fluid strongly reacted with early meconium and fetal small intestine in immunodiffusion, enzyme immunoassay, and staining with immunoperoxidase. The results suggest that most of the enzyme rich particulate fraction in early amniotic fluid originates in the fetal intestine.
Asunto(s)
Líquido Amniótico/enzimología , Fosfatasa Alcalina/metabolismo , Líquido Amniótico/citología , Líquido Amniótico/inmunología , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Leucil Aminopeptidasa/metabolismo , Meconio/inmunología , Microscopía Electrónica , Microvellosidades/enzimología , Embarazo , Segundo Trimestre del Embarazo , Sacarasa/metabolismo , Trehalasa/metabolismo , gamma-Glutamiltransferasa/metabolismoRESUMEN
The success of transplantation of human embryonic mesencephalic tissue to treat parkinsonian patients is limited by the poor survival of the transplant. We show that an AAV2 vector mediates efficient expression of the egfp reporter gene in organotypic cultures of freshly explanted solid fragments of rat embryonic ventral mesencephalon (VM). We observed early and sustained transgene expression (4 days to > or = 6 weeks). Furthermore, rAAV-infected rat embryonic VM transplanted in the adult striatum continued to express EGFP for > or = 3 months. More than 95% of the transduced cells were neurons. Dopaminergic neurons were transduced at low frequency at earlier time points. This method of gene delivery could prove useful to achieve local, continuous secretion of neurotrophic factors at physiologically relevant doses to treat Parkinson's disease.
Asunto(s)
Trasplante de Tejido Encefálico , Trasplante de Tejido Fetal , Trastornos Parkinsonianos/cirugía , Animales , Dopamina/fisiología , Expresión Génica/fisiología , Vectores Genéticos , Supervivencia de Injerto/fisiología , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Masculino , Mesencéfalo/trasplante , Degeneración Nerviosa/fisiopatología , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
The success of transplantation of human fetal mesencephalic tissue into the putamen of patients with Parkinson's disease (PD) is still limited by the poor survival of the graft. In animal models of fetal transplantation for PD, antiapoptotic agents, such as growth factors or caspase inhibitors, or agents counteracting oxidative stress enhance the survival and reinnervation potential of the graft. Genetic modification of the transplant could allow a local and continuous delivery of these factors at physiologically relevant doses. The major challenge remains the development of strategies to achieve both early and sustained gene delivery in the absence of vector-mediated toxicity. We recently reported that E14 rat fetal mesencephalon could be efficiently tranduced by adeno-associated virus type 2 (AAV2) vectors and that gene expression was maintained until at least 3 months after transplantation in the adult rat striatum. Here we report that an AAV2 vector can mediate the expression of the EGFP reporter gene under the control of a CMV promoter in organotypic cultures of freshly explanted solid fragments of human fetal mesencephalic tissue as early as 3 days to at least 6 weeks postinfection. These results suggest that AAV2 vectors could be used to genetically modify the human fetal tissue prior to transplantation to Parkinson's patients to promote graft survival and integration.
Asunto(s)
Trasplante de Tejido Encefálico/métodos , Trasplante de Células/métodos , Dependovirus/genética , Trasplante de Tejido Fetal/métodos , Técnicas de Transferencia de Gen , Vectores Genéticos , Mesencéfalo/citología , Animales , Medios de Cultivo , Citomegalovirus/genética , Regulación de la Expresión Génica , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/genética , Humanos , Enfermedad de Parkinson/terapia , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Transducción GenéticaRESUMEN
The dental papilla cells play a major regulatory role during tooth morphogenesis, and they are the only mesenchymal cells capable of differentiating into odontoblasts secreting dentin. In this paper, we have extended our studies on the behavior of cultured dental papilla cells which have been disaggregated from 17-day mouse embryo teeth. Quite unexpectedly, we observed that these cells, which in vivo are embedded in a fibronectin-rich extracellular matrix, lose all surface-associated fibronectin when cultured as monolayers. Fibronectin was, however, detected intracellularly, and metabolic labeling and immunoprecipitation studies indicated that the dental papilla cells continued to synthesize fibronectin in culture. Furthermore, when purified plasma fibronectin was added at 50 micrograms/mL to the culture medium, it became incorporated as fibrillar matrix on the surfaces of dental papilla cells. This indicates that the cells are not deficient in cell-surface receptors or other surface-associated molecules which bind fibronectin. When pieces of dental papillae were cultured as explants, an abundant matrix containing fibronectin was deposited on their surfaces. This matrix was gradually lost as the cells migrated from the explants. Furthermore, when the cells were disaggregated and cultured at high cell density, the cells in the central area of the pellet were covered by fibronectin containing fibrillar structures which were lost as the cells spread out. This indicates that the maintenance of close contacts between the dental papilla cells is required for the assembly of fibronectin into the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Papila Dental/citología , Matriz Extracelular/metabolismo , Fibronectinas/biosíntesis , Germen Dentario/citología , Animales , Células Cultivadas , Papila Dental/metabolismo , Papila Dental/ultraestructura , Embrión de Mamíferos , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Encía/citología , Encía/ultraestructura , Mesodermo/citología , Mesodermo/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía ElectrónicaRESUMEN
The molecular specificity of the dental papilla of a bell-stage tooth was studied by production of dental-papilla-reactive monoclonal antibodies (Mabs). One of the Mabs, designated 7C5, recognized an epitope present in glycosaminoglycan. Several lines of evidence suggested that the 7C5-epitope consists of chondroitin 6-sulfate. The Mab did not react with mouse dental epithelium, but reacted uniformly with mesenchymal tissue in the mandibular process and accumulated in the dental sac and in the papilla of bell-stage tooth germs. The 7C5-staining was lost from the differentiating odontoblasts, while the staining in the molar tooth papilla was accumulated in the subodontoblastic layer. In the developing mouse incisor, the 7C5-epitope was restricted to the lingual-posterior area. The 7C5-epitope was also present in pulpal tissue and predentin of different types of teeth of various mammalian species, including man, sheep, swine, and rat. Collagenase pre-treatment of tissue sections abolished the bulk of the 7C5-reactivity in peridental mesenchyme during embryonic stages while leaving the staining of the dental papilla intact. In newborn and adult teeth, collagenase also impaired the reactivity in the pulp except for the subodontoblastic layer. This suggests the existence of different subpopulations of the 7C5-epitope containing proteoglycans in dental papilla and pulp. A high-molecular-weight proteoglycan, sensitive to chondroitinase ABC but not to heparinase or heparitinase, was immunoprecipitated by 7C5 from extracts of bell-stage mouse tooth germs. We suggest that the evolutionary conservation of chondroitin 6-sulfate in the dental pulp reflects its properties as non-terminally differentiated tissue and perhaps the retention of a potential to differentiate to odontoblasts.