RESUMEN
OBJECT: The presence of a "July effect," where the influx of new residents and fellows at teaching hospitals every July may negatively affect patient care and outcomes, is widely debated. The authors used the Nationwide Inpatient Sample (NIS) to identify all cases of spinal surgery and examine outcomes among patients who underwent surgery in July compared with those who underwent surgery in other months. METHODS: Spinal surgery hospitalizations from 2001 to 2008 were identified in the NIS by extracting relevant ICD-9 codes. Rates of in-hospital mortality, discharge to a long-term care facility, and postoperative complications were compared between admission months and between teaching and nonteaching hospitals using the Wilcoxon rank-sum test, Fisher exact test, and multivariate regression analysis. RESULTS: Compared with patients admitted in other months, patients who were admitted to teaching hospitals in July for spinal surgery showed a similar likelihood of in-hospital mortality (OR 0.94 [95% CI 0.78-1.11], p = 0.46), reaction to implanted device/instrumentation (OR 0.88 [95% CI 0.77-1.02], p = 0.09), and postoperative wound dehiscence (OR 1.12 [95% CI 0.94-1.33], p = 0.25). A significantly higher likelihood of discharge to a long-term care facility (OR 1.03 [95% CI 1.00-1.07], p = 0.0467) and postoperative infection (OR 1.11 [95% CI 1.05-1.17], p = 0.0341) was observed in teaching hospitals in July compared with other months; however, incidence rates were similar regardless of admission month. Higher-risk patients (Charlson score ≥ 2) admitted to teaching hospitals in July had a similar likelihood of all outcomes regardless of admission month. CONCLUSIONS: This study of nationwide hospitalizations demonstrates that the influx of new residents and fellows in July has a negligible effect on periprocedural outcomes following spinal surgery.
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Hospitales de Enseñanza , Complicaciones Posoperatorias/epidemiología , Estaciones del Año , Enfermedades de la Columna Vertebral/cirugía , Adulto , Anciano , Comorbilidad , Femenino , Precios de Hospital , Mortalidad Hospitalaria , Humanos , Incidencia , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Admisión del Paciente/estadística & datos numéricos , Complicaciones Posoperatorias/mortalidad , Enfermedades de la Columna Vertebral/mortalidad , Resultado del Tratamiento , Estados Unidos/epidemiologíaRESUMEN
Mechanical loading has been described as a highly important stimulus for improvements in the quality and strength of bone. It has also been shown that mechanical stimuli can induce the differentiation of mesenchymal stem cells (MSCs) along the osteogenic lineage. We have previously demonstrated the potent osteogenic effect of MSCs engineered to overexpress the BMP2 gene. In this study we investigated the effect of mechanical loading on BMP2-expressing MSC-like cells, using a special bioreactor designed to apply dynamic forces on cell-seeded hydrogels. Cell viability, alkaline phosphatase (ALP) activity, BMP2 secretion and mineralized substance formation in the hydrogels were quantified. We found that cell metabolism increased as high as 6.8-fold, ALP activity by 12.5-fold, BMP2 secretion by 182-fold and mineralized tissue formation by 1.72-fold in hydrogels containing MSC-like cells expressing BMP2, which were cultured in the presence of mechanical loading. We have shown that ex vivo mechanical loading had an additive effect on BMP2-induced osteogenesis in genetically engineered MSC-like cells. These data could be valuable for bone tissue-engineering strategies of the future.
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Ingeniería Genética/métodos , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Fosfatasa Alcalina/metabolismo , Animales , Reactores Biológicos , Proteína Morfogenética Ósea 2/metabolismo , Huesos/metabolismo , Diferenciación Celular , Fibrinógeno/química , Hidrogeles , Ratones , Ratones Endogámicos C3H , Osteogénesis , Polietilenglicoles/químicaRESUMEN
OBJECTIVE: Meningiomas are the second most common primary tumors of the central nervous system. Meningiomas at the cranial base pose technical challenges and result in increased morbidity. To investigate the molecular mechanisms of meningioma formation, the expression profiles of 12 000 genes from meningiomas and dural specimens were compared. METHODS: Ribonucleic acid from 6 meningiomas (World Health Organization Grade I) and 4 dural specimens was profiled using U95A GeneChips (Affymetrix, Inc., Santa Clara, CA). Expression profiles of the 2 groups were compared using dChip and Data Mining Tool software packages (Affymetrix, Inc.) to identify differentially expressed genes. Down-regulation of a differentially expressed tumor suppressor gene, deleted in liver cancer 1 (DLC1), was verified by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Function and methylation of DLC1 were assessed by ectopic expression in 5 primary cultures, demethylation assay using 5-aza-2'-deoxycytidine, and methylation-specific polymerase chain reaction in 4 meningioma samples. RESULTS: Gene expression profiling revealed up-regulation of 5 genes (fibroblast growth factor 9, gibbon leukemia virus receptor 2, cyclin D1, eukaryotic translation initiation factor 5A, and 28S ribosomal ribonucleic acid) and down-regulation of 35 genes, including DLC1, in meningiomas. The down-regulation of DLC1 in meningiomas was confirmed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Transfection of DLC1 complementary deoxyribonucleic acid into primary cultures of 5 meningiomas resulted in decreased replication. Although demethylation decreased meningioma cell growth rates in vitro, methylation-specific polymerase chain reaction did not detect DLC1 promoter methylation. CONCLUSION: The results suggest that DLC1 may function as a tumor suppressor gene in meningiomas. Furthermore, DLC1 promoter methylation does not appear to be responsible for the decreased DLC1 expression in these tumors.
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Meningioma/genética , Proteínas Supresoras de Tumor/genética , Adenoviridae/genética , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular , Metilación de ADN/genética , Regulación hacia Abajo , Duramadre/metabolismo , Femenino , Proteínas Activadoras de GTPasa , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Masculino , Meningioma/metabolismo , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Meningiomas and schwannomas are the two most common extra-axial intracranial tumors in adults. Since their initial discovery, these often-benign lesions have shared a parallel metamorphosis in their management. The goal of this article is to provide a review of the current literature surrounding the mainstays of therapy for these lesions.
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Neoplasias Encefálicas/terapia , Neoplasias Meníngeas/terapia , Meningioma/terapia , Neurilemoma/terapia , Humanos , Microcirugia , Procedimientos NeuroquirúrgicosRESUMEN
We report a fatal case of brain abscess caused by infection due to a community-associated methicillin-resistant Staphylococcus aureus strain (USA300) in a 37-year-old incarcerated woman with a history of furunculosis and injection drug use. Community-onset pyogenic brain abscess should be added to the growing list of life-threatening invasive infections caused by epidemic community-acquired methicillin-resistant S. aureus.
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Absceso Encefálico/microbiología , Infecciones Comunitarias Adquiridas/microbiología , Resistencia a la Meticilina , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Adulto , Absceso Encefálico/diagnóstico , Infecciones Comunitarias Adquiridas/diagnóstico , Resultado Fatal , Femenino , Humanos , Prisioneros , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/diagnóstico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Many clinical conditions require regeneration or implantation of bone. This is one focus shared by neurosurgery and orthopedics. Current therapeutic options (bone grafting and protein-based therapy) do not provide satisfying solutions to the problem of massive bone defects. In the past few years, gene- and stem cell-based therapy has been extensively studied to achieve a viable alternative to current solutions offered by modern medicine for bone-loss repair. The use of adult stem cells for bone regeneration has gained much focus. This unique population of multipotential cells has been isolated from various sources, including bone marrow, adipose, and muscle tissues. Genetic engineering of adult stem cells with potent osteogenic genes has led to fracture repair and rapid bone formation in vivo. It is hypothesized that these genetically modified cells exert both an autocrine and a paracrine effects on host stem cells, leading to an enhanced osteogenic effect. The use of direct gene delivery has also shown much promise for in vivo bone repair. Several viral and nonviral methods have been used to achieve substantial bone tissue formation in various sites in animal models. To advance these platforms to the clinical setting, it will be mandatory to overcome specific hurdles, such as control over transgene expression, viral vector toxicity, and prolonged culture periods of therapeutic stem cells. This review covers a prospect of cell and gene therapy for bone repair as well as some very recent advancements in stem cell isolation, genetic engineering, and exogenous control of transgene expression.
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Enfermedades Óseas/genética , Enfermedades Óseas/terapia , Regeneración Ósea/genética , Terapia Genética/tendencias , Osteogénesis/genética , Trasplante de Células Madre/tendencias , Ingeniería de Tejidos/tendencias , Animales , HumanosRESUMEN
Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assess the effectiveness and safety of systems of gene therapy delivery. In the present study, we employed a CCD (charge-coupled-device) imaging system to monitor how a prostate-specific adenovirus vector (AdPSA-Luc) mediated the long-term, sustained expression of firefly luciferase (Luc) in living human prostate cancer mouse models. The in vivo bioluminescence imaging revealed significantly high levels of luciferase expression up to 1 month, not only in prostate tumours, but also in lungs after intratumoural injection. Systemic tail vein injection of AdPSA-Luc revealed significant luciferase expression in lungs of both human prostate cancer mouse models and naïve mice, but significantly higher in the former, while the control virus, AdCMV-Luc, containing CMV (cytomegalovirus) promoter and luciferase gene, just restricted expression in the livers. Our findings demonstrate the ability of the cooled CCD camera to sensitively and non-invasively track the location, magnitude and persistence of luciferase gene expression in human prostate cancer mouse models. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.
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Diagnóstico por Imagen , Genes Reporteros , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Neoplasias de la Próstata/diagnóstico , Animales , Humanos , Proteínas Luminiscentes/metabolismo , Neoplasias Pulmonares/diagnóstico , Masculino , Ratones , Ratones Desnudos , Neoplasias Primarias Múltiples/diagnóstico , Neoplasias de la Próstata/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND PURPOSE: The purpose of this study was to determine whether implanting exogenous fibroblasts on platinum coils could enhance intra-aneurysmal fibrosis. Hypotheses included: (1) fibroblast-coated (FBC) platinum coils can improve angiographic results after embolization; and (2) FBC platinum coils can accelerate histological healing of embolized aneurysms. METHODS: Experimental aneurysms in rabbits were embolized with control platinum coils (n=18) or FBC coils (n=18). Subjects were euthanized at 14 days, 1 month, 3 months and 6 months after implantation. Digital subtraction angiography was used to evaluate stability after embolization. Histological samples were examined with a grading system (range, 0 to 12) based on neck and dome healing. RESULTS: Histology total scores and fibrosis ratio at 14 days were significantly greater in the FBC coil group compared with controls (6.6+/-1.9 versus 2.5+/-1.1, 1.2+/-0.6% versus 0.2+/-0.3%, respectively; P=0.0090). Cavities embolized with FBC coils showed cellular proliferation and thrombus organization, with an endothelialized membrane bridging the neck. There were no differences between groups in the later timepoints. The FBC coil group showed radiographic stability in 11 (61%) cases, coil compaction in 2 (11%) cases, and progressive occlusion in 5 (28%) cases. No progressive occlusion was seen in controls; 3 (17%) of 18 control cases exhibited coil compaction (P=0.0546). CONCLUSIONS: FBC coils can accelerate early histological healing compared with control coils in the rabbit aneurysm model.
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Embolización Terapéutica/instrumentación , Embolización Terapéutica/métodos , Fibroblastos/trasplante , Aneurisma Intracraneal/terapia , Prótesis e Implantes/tendencias , Actinas/metabolismo , Animales , Biomarcadores , Células Cultivadas , Angiografía Cerebral , Modelos Animales de Enfermedad , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Aneurisma Intracraneal/diagnóstico por imagen , Aneurisma Intracraneal/patología , Masculino , Mioblastos del Músculo Liso , Platino (Metal)/uso terapéutico , Prótesis e Implantes/normas , Conejos , Resultado del Tratamiento , Vimentina/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND: Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit marked differences in atherosclerosis susceptibility. We sought to determine whether the difference in atherosclerosis susceptibility resides at the level of arterial walls. METHODS AND RESULTS: Thoracic aortic segments from 8-week-old female B6 and C3H apolipoprotein E-deficient mice were transplanted into the infrarenal aorta of 10-week-old female F1 mice. After transplantation, recipients were maintained on a chow diet for 16 weeks. The donor aortic segments of B6 mice developed significantly larger atherosclerotic lesions than those of C3H (44,983+/-11,702 versus 5600+/-4885 microm2 per section; P=0.011). Expression of vascular cell adhesion molecule (VCAM)-1 by endothelial cells was examined both in vitro and in vivo. B6 mice expressed significantly more VCAM-1 than their C3H counterparts. Sequence analysis of VCAM-1 cDNA revealed a nucleotide difference in the coding region that resulted in substitution of an amino acid in the protein product. CONCLUSIONS: These data provide direct proof that factors operating in the vessel wall, particularly endothelial cells, can serve as atherosclerosis modifiers and suggest a possibility for the contribution of VCAM-1 to atherosclerosis susceptibility.
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Aorta Torácica/anatomía & histología , Aorta Torácica/trasplante , Aterosclerosis/genética , Molécula 1 de Adhesión Celular Vascular/genética , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Lípidos/sangre , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante HomólogoRESUMEN
OBJECT: Promotion of the repair and regeneration of damaged adult neurons is a major goal of neurological science. In this study, the effects of G protein-coupled receptor kinase interacting protein 1 (GIT1) overexpression in human neuron cells were tested in human neuronal cells by using an adenoviral vector. METHODS: A recombinant GIT1 and enhanced green fluorescent protein (EGFP) adenoviral vector (AdGIT1) was created by using a standard viral construction procedure. Human neuronal (NT2N) cells, which had been derived from an NT2 human teratocarcinoma cell line, were used in this experiment. Immunocytochemical methods were applied to identify NT2N cells with neural features and to probe the relationship among signaling proteins. Several biological activities were assessed, including neural spine formation, cell migration, and the levels of expression of growth-associated protein-43 (GAP-43) and active Cdc42. The number of cells with spine formation and the number of migrated cells were significantly higher in the AdGIT1-treated group of NT2N cells than in untreated (control) NT2N cells or in AdEGFP-treated NT2N cells. The levels of GAP-43 and active Cdc42 expression were significantly higher in the AdGITl-treated group than that in the other two cell groups. CONCLUSIONS: The results of this study demonstrate that GIT1 overexpression has the potential to promote neural spine formation and cell migration in human neuronal cells. At the same time, the increased level of GAP-43 in GIT1-overexpressed cells indicates that GIT1 may have the potential to improve growth and regeneration of damaged axons. The GIT1-beta-PIX-Cdc42-PAK pathway may play an important role in neuronal outgrowth.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de Ciclo Celular/fisiología , Neuronas/fisiología , Adenoviridae , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular , Proteína GAP-43/metabolismo , Vectores Genéticos , Humanos , Organogénesis , Médula Espinal/embriología , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Osteogenic potentials of some recombinant human bone morphogenetic protein (BMP) first-generation adenoviral vectors (ADhBMPs) are significantly limited in immunocompetent animals. It is unclear what role expression of viral proteins and foreign proteins transduced by adenoviral vectors play in the host immune response and in ectopic bone formation. In this study two sets of experiments were designed and performed. First, rat BMP6 cDNA were amplified, sequenced, and recombined in first-generation adenoviral vector (ADrBMP6). A comparison of human and rat BMP6 adenoviral vectors demonstrated identical osteogenic activities in both immunodeficient and immunocompetent rats. Second, the activities of recombinant human BMP6 in E1- (ADhBMP6) and [E1-,E2b-] ( [E1-,E2b-]ADGFP&hBMP6, and [E1-,E2b-]ADhBMP6) adenoviral vectors were compared in both in vitro and in vivo models. Similar activities of these two generations of BMP adenoviral vectors were found in all models. These results indicate that the amount of viral gene expression and the source of the BMP cDNA are not major factors in the interruption of osteogenic potentials of recombinant BMP6 adenoviral vectors in immunocompetent animals.
RESUMEN
There are several gene therapy approaches to tissue regeneration. Although usually efficient, virusbased approaches may elicit an immune response against the viral proteins. An alternative approach, nonviral transfer, is safer, and can be controlled and reproduced. We hypothesized that in vivo bone formation could be achieved using human mesenchymal stem cells (hMSCs) nonvirally transfected with the human bone morphogenetic protein-2 (hBMP-2) or -9 (hBMP-9) gene. Human MSCs were transfected using nucleofection, a unique electropermeabilization-based technique. Postnucleofection, cell viability was 53.6 +/- 2.5% and gene delivery efficiency was 51% to 88% (mean 68.2 +/- 4.1%), as demonstrated by flow cytometry in enhanced green fluorescent protein (EGFP)-nucleofected hMSCs. Transgene expression lasted longer than 14 days and was very low 21 days postnucleofection. Both hBMP-2- and hBMP-9-nucleofected hMSCs in culture demonstrated a significant increase in calcium deposition compared with EGFP-nucleofected hMSCs. Human BMP-2- and hBMP-9-nucleofected hMSCs transplanted in ectopic sites in NOD/SCID mice induced bone formation 4 weeks postinjection. We conclude that in vivo bone formation can be achieved by using nonvirally nucleofected hMSCs. This could lead to a breakthrough in the field of regenerative medicine, in which safer, nonviral therapeutic strategies present a very attractive alternative.
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Proteínas Morfogenéticas Óseas/genética , Regeneración Ósea/fisiología , Terapia Genética , Células Madre Mesenquimatosas/metabolismo , Ingeniería de Tejidos/métodos , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Regeneración Ósea/genética , Calcio/análisis , Supervivencia Celular , Células Cultivadas , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/metabolismo , Factor 2 de Diferenciación de Crecimiento , Factores de Diferenciación de Crecimiento , Humanos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Transgenes , Trasplante HeterólogoRESUMEN
LDL receptor-deficient (LDLR(-/-)) mice exhibit mild hyperlipidemia on a chow diet but develop severe hyperlipidemia on a high fat diet. In this study, we investigated neointimal formation after removal of the endothelium when LDLR(-/-) mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 10 weeks of age, female mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, neointimal formation was barely detectable in the injured vessel when mice developed mild hyperlipidemia on the chow diet. In contrast, neointimal lesions were obvious when mice developed severe hyperlipidemia on the Western diet. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells and smooth muscle cells in neointimal lesions. The injured artery also exhibited a significant increase in medial area on the Western diet. Plasma levels of MCP-1 and soluble VCAM-1 were significantly elevated by feeding of the Western diet. These data indicate that hyperlipidemia aggravates neointimal growth in LDLR(-/-) mice by promoting foam cell formation and inflammation.
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Hiperlipidemias/genética , Hiperlipidemias/patología , Receptores de LDL/genética , Receptores de LDL/fisiología , Túnica Íntima/patología , Alimentación Animal , Animales , Quimiocina CCL2/biosíntesis , Femenino , Células Espumosas/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
Dyslipidemia and hyperglycemia are integral components of the metabolic perturbations in type 2 diabetes. Apolipoprotein E-deficient (apoE(-/-)) mice develop severe hyperlipidemia and significant hyperglycemia when fed a western diet containing 21% fat (w/w), 0.15% cholesterol and 19.5% casein. Using an intercross between C57BL/6J (B6) and C3H/HeJ (C3H) apoE(-/-) mice, we performed quantitative trait locus (QTL) analysis to identify loci contributing to hyperglycemia and associated traits. Fasting plasma levels of glucose, insulin and serum amyloid-P (SAP) and body weight in 234 female F2 mice were measured after being fed the western diet for 12 weeks. QTL analysis revealed one significant QTL, named Bglu3 [95.8 cM, logarithm of odds ratio (OR)(LOD) 4.1], on chromosome 1 and a suggestive QTL on chromosome 9 (38 cM, LOD 2.3) that influenced plasma glucose levels. Bglu3 coincided with loci on distal chromosomal 1 that had a major influence on plasma SAP levels and body weight. Significant correlations between plasma glucose, SAP and body weight were observed in F2 mice. Thus, these results demonstrate genetic linkages of hyperglycemia and body weight with SAP, a marker of the acute-phase response, in hyperlipidemic apoE(-/-) mice and suggest a probability for the Sap gene to be a positional candidate of Bglu3.
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Apolipoproteínas E/metabolismo , Peso Corporal/fisiología , Ligamiento Genético , Hiperglucemia/genética , Hiperglucemia/patología , Componente Amiloide P Sérico/metabolismo , Animales , Apolipoproteínas E/genética , Glucemia/metabolismo , Peso Corporal/genética , Cromosomas de los Mamíferos/genética , Cruzamientos Genéticos , Femenino , Alimentos Formulados , Insulina/sangre , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Carácter Cuantitativo HeredableRESUMEN
The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.
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Apolipoproteínas E/deficiencia , Aterosclerosis/enzimología , Aterosclerosis/metabolismo , Óxido Nítrico Sintasa de Tipo II/deficiencia , Animales , Aorta/citología , Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerosis/genética , Células Cultivadas , LDL-Colesterol/sangre , LDL-Colesterol/metabolismo , Cruzamientos Genéticos , Femenino , Heterocigoto , Peroxidación de Lípido , Peróxidos Lipídicos/sangre , Peróxidos Lipídicos/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintasa de Tipo II/genéticaRESUMEN
The osteogenic potential of AAV5hBMP6 was compared with that of ADhBMP6 in immunodeficient and immunocompetent rats. AAV5hBMP6 (2.3 x 10(12) particles) and ADhBMP6 (5 x 10(7) PFU) elicited viral antibody production in immunocompetent rats. Among rats that received AAV5hBMP6, the earliest time points at which the bone was visible under CT scanner were 30 days in 2-month-old Sprague-Dawley (SD) rats and 60 days in 18-month-old SD rats. The mean volumes of ectopic bone 90 days after viral injection were 0.31 +/- 0.14 cm(3) in athymic nude rats, 0.64 +/- 0.12 cm(3) in 2-month-old SD rats, and 0.21 +/- 0.10 cm(3) in 18-month-old SD rats. In contrast, among rats that received ADhBMP6, the earliest time points to observe the bone formation by CT scan were 15 days in 2-month-old rats and no bone formation in 18-month-old SD rats. The mean volumes of ectopic bone were 4.17 +/- 0.05 cm(3) in athymic nude rats and 0.06 +/- 0.03 cm(3) in 2-month-old SD rats. Although both types of viruses induced an immune response in immunocompetent animals, this response played different roles in the process of bone formation induced by the BMP6 vectors.
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Adenoviridae/genética , Proteínas Morfogenéticas Óseas/farmacología , Dependovirus/genética , Osteogénesis/fisiología , Animales , Proteína Morfogenética Ósea 6 , Proteínas Morfogenéticas Óseas/administración & dosificación , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Vectores Genéticos , Humanos , Músculo Esquelético/citología , Osteogénesis/efectos de los fármacos , Ratas , Ratas Desnudas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Especificidad de la Especie , Factores de Tiempo , Tomografía por Rayos XRESUMEN
Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) differ significantly in atherosclerosis susceptibility and plasma lipid levels on the apolipoprotein E-deficient (apoE-/-) background when fed a Western diet. To determine genetic factors contributing to the variations in these phenotypes, we performed quantitative trait locus (QTL) analysis using an intercross between the two strains carrying the apoE-/- gene. Atherosclerotic lesions at the aortic root and plasma lipid levels of 234 female F2 mice were analyzed after being fed a Western diet for 12 weeks. QTL analysis revealed one significant QTL, named Ath22 (42 cM, LOD 4.1), on chromosome 9 and a suggestive QTL near D11mit236 (20 cM, LOD 2.4) on chromosome 11 that influenced atherosclerotic lesion size. One significant QTL on distal chromosome 1, which accounted for major variations in plasma LDL/VLDL cholesterol and triglyceride levels, coincided with a QTL having strong effects on body weight. Plasma LDL/VLDL cholesterol or triglyceride levels of F2 mice were significantly correlated with body weight, but they were not correlated with atherosclerotic lesion sizes. These data indicate that atherosclerosis susceptibility and plasma cholesterol levels are controlled by separate genetic factors in the B6 and C3H mouse model and that genetic linkages exist between body weight and lipoprotein metabolism.
Asunto(s)
Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Cruzamientos Genéticos , Predisposición Genética a la Enfermedad , Sitios de Carácter Cuantitativo , Animales , Aterosclerosis/patología , Peso Corporal/genética , LDL-Colesterol/sangre , LDL-Colesterol/genética , VLDL-Colesterol/sangre , VLDL-Colesterol/genética , Femenino , Lipoproteínas HDL/sangre , Lipoproteínas HDL/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Triglicéridos/sangre , Triglicéridos/genéticaRESUMEN
OBJECT: The authors hypothesized that spinal fusion can be achieved and monitored by using cell-mediated gene therapy. Mesenchymal stem cells (MSCs) genetically engineered to express recombinant human bone morphogenetic protein-2 (rhBMP-2) conditionally, were implanted into the paraspinal muscles of mice to establish spinal fusion. The goal was to demonstrate an MSC-based gene therapy platform in which controlled gene expression is used to obtain spinal fusion in a murine model. METHODS: Mesenchymal stem cells expressing the rhBMP-2 gene were injected into the paravertebral muscle in mice. Bone formation in the paraspinal region was longitudinally followed by performing micro-computerized tomography scanning, histological studies, and an analysis of osteocalcin expression to demonstrate the presence of engrafted engineered MSCs. The minimal period of rhBMP-2 expression by the engineered MSCs required to induce fusion was determined. The results of this study demonstrate that genetically engineered MSCs induce bone formation in areas adjacent to and touching the posterior elements of the spine. This newly formed bone fuses the spine, as demonstrated by radiological and histological studies. The authors demonstrate that injected cells induce active osteogenesis at the site of implantation for up to 4 weeks postinjection. They found that a 7-day induction of rhBMP-2 expression in genetically engineered MSCs was sufficient to form new bone tissue, although the quantity of this bone increased as longer expression periods were implemented. CONCLUSIONS: After their injection genetically engineered MSCs can efficiently form new bone in the paraspinal muscle of the mouse to obtain spinal fusion. The extent and quantity of this newly formed bone can be monitored by controlling the duration of rhBMP-2 gene expression.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Ingeniería Genética , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Osteogénesis/fisiología , Fusión Vertebral/métodos , Factor de Crecimiento Transformador beta/fisiología , Animales , Proteína Morfogenética Ósea 2 , Estudios de Factibilidad , Femenino , Ratones , Ratones Endogámicos C3H , Modelos Animales , Factores de TiempoRESUMEN
PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10(9)PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.
Asunto(s)
Luciferasas de Luciérnaga/metabolismo , Pulmón/metabolismo , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Genes Reporteros/genética , Humanos , Luciferasas de Luciérnaga/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
Recruitment of inflammatory cells in the arterial wall by vascular adhesion molecules plays a key role in development of atherosclerosis. Apolipoprotein E-deficient (apoE(-/-)) mice have spontaneous hyperlipidemia and develop all phases of atherosclerotic lesions. We sought to examine plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and sP-selectin in two apoE(-/-) strains C57BL/6 (B6) and BALB/c with early or advanced lesions. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. On either diet, BALB/c.apoE(-/-) mice developed much smaller atherosclerotic lesions and displayed significantly lower levels of sVCAM-1 and sP-selectin than B6.apoE(-/-) mice. The Western diet significantly elevated sVCAM-1 levels in both strains and sP-selectin levels in B6.apoE(-/-) mice. BALB/c.apoE(-/-) mice exhibited 2-fold higher HDL cholesterol levels on the chow diet and 15-fold higher HDL levels on the Western diet than B6.apoE(-/-) mice, although the two strains had comparable levels of total cholesterol and triglyceride. Thus, increased atherosclerosis is accompanied by increases in circulating VCAM-1 and P-selectin levels in the two apoE(-/-) mouse strains, and the high HDL level may protect against atherosclerosis by inhibiting the expression of adhesion molecules in BALB/c.apoE(-/-) mice.