RESUMEN
BACKGROUND: The inherent toxicity of unmodified Quantum Dots (QDs) is a major hindrance to their use in biological applications. To make them more potent as neuroprosthetic and neurotherapeutic agents, thioglycolic acid (TGA) capped CdTe QDs, were coated with a gelatine layer and investigated in this study with differentiated pheochromocytoma 12 (PC12) cells. The QD--cell interactions were investigated after incubation periods of up to 17 days by MTT and APOTOX-Glo Triplex assays along with using confocal microscopy. RESULTS: Long term exposure (up to 17 days) to gelatinated TGA-capped CdTe QDs of PC12 cells in the course of differentiation and after neurites were grown resulted in dramatically reduced cytotoxicity compared to non-gelatinated TGA-capped CdTe QDs. CONCLUSION: The toxicity mechanism of QDs was identified as caspase-mediated apoptosis as a result of cadmium leaking from the core of QDs. It was therefore concluded that the gelatine capping on the surface of QDs acts as a barrier towards the leaking of toxic ions from the core QDs in the long term (up to 17 days).
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos de Cadmio/toxicidad , Gelatina/farmacología , Puntos Cuánticos , Telurio/toxicidad , Animales , Compuestos de Cadmio/química , Compuestos de Cadmio/farmacocinética , Diferenciación Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Gelatina/química , Neuritas/efectos de los fármacos , Células PC12 , Ratas , Telurio/química , Telurio/farmacocinética , Tioglicolatos/química , Pruebas de Toxicidad CrónicaRESUMEN
BACKGROUND: Although indexed left atrial volume (iLAV) is the most accurate measure of left atrial size, it has not been evaluated prospectively as predictor of recurrence of atrial fibrillation (AFib) after successful cardioversion (CV). METHODS: We prospectively selected 76 patients (mean age 66.1 ± 13.6 years, 65.8% men) with AFib who underwent successful CV. Baseline clinical and echocardiographic characteristics were obtained before CV. LAV was measured using Simpson's method and indexed to body surface area. All patients were scheduled for follow-up visit at 1, 6, 12 months, and then annually. A 24-hour Holter ECG was performed within 6 months and each time the patients reported symptoms suggestive of arrhythmia. RESULTS: The 52 patients (68.4%) with AFib recurrence had larger iLAV (35.5 ± 8.9 mL/m(2) vs 27.0 ± 6.7 mL/m(2) , P < 0.001). Anteroposterior LA diameter was not associated with AFib relapse (OR 1.08, 95% CI: 0.96-1.21, P = 0.09). Each unit increase in iLAV was associated with a 1.15-fold increased risk of recurrence (OR 1.15, 95% CI: 1.06-1.25, P < 0.001). In a multivariable model, iLAV remained the only independent predictor of relapse (adjusted OR 1.14, 95% CI: 1.02-1.28, P = 0.02). The area under ROC curves, generated to compare LA diameter, and iLAV as predictors of AFib recurrence were 0.56 (SE 0.07) versus 0.78 (SE 0.05), respectively (P = 0.003). CONCLUSION: This is the first prospective study to show that larger iLAV, as a more accurate measure of LA remodeling than anteroposterior diameter, is strongly and independently associated with a higher risk of AFib recurrence after CV.
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Fibrilación Atrial/diagnóstico por imagen , Fibrilación Atrial/prevención & control , Ecocardiografía Tridimensional/métodos , Atrios Cardíacos/diagnóstico por imagen , Anciano , Femenino , Enfermedades de las Válvulas Cardíacas , Humanos , Masculino , Tamaño de los Órganos , Pronóstico , Reproducibilidad de los Resultados , Prevención Secundaria , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
BACKGROUND: The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/ß-glycerol phosphate (ß-GP) as a carrier. METHODS: Three chitosan/ß-GP scaffolds were prepared with varying concentrations of chitosan and ß-GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin-3 (Lenti NT-3) was incorporated into the chitosan/ß-GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of pre-seeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence-activated cell sorting analysis. The effect of Lenti NT-3 was analyzed by measuring DRG neurite outgrowth. RESULTS: Collagen displayed its most significant elution of virus on day 1 and chitosan/ß-GP (with a final concentration of 2.17% chitosan) on day 3. CONCLUSIONS: The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long-term gene expression for therapeutic factors to treat conditions such as injury to the nervous system.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos/metabolismo , Hidrogeles/química , Neurotrofina 3/metabolismo , Animales , Quitosano/química , Colágeno/química , Medios de Cultivo/química , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Citometría de Flujo , Ganglios Espinales/citología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Glicerofosfatos/química , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Lentivirus/genética , Lentivirus/metabolismo , Neuritas/metabolismo , Ratas , Ratas Sprague-Dawley , Temperatura , Factores de Tiempo , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
BACKGROUND: The unique and tuneable photonic properties of Quantum Dots (QDs) have made them potentially useful tools for imaging biological entities. However, QDs though attractive diagnostic and therapeutic tools, have a major disadvantage due to their inherent cytotoxic nature. The cellular interaction, uptake and resultant toxic influence of CdTe QDs (gelatinised and non-gelatinised Thioglycolic acid (TGA) capped) have been investigated with pheochromocytoma 12 (PC12) cells. In conjunction to their analysis by confocal microscopy, the QD - cell interplay was explored as the QD concentrations were varied over extended (up to 72 hours) co-incubation times. Coupled to this investigation, cell viability, DNA quantification and cell proliferation assays were also performed to compare and contrast the various factors leading to cell stress and ultimately death. RESULTS: Thioglycolic acid (TGA) stabilised CdTe QDs (gel and non - gel) were co-incubated with PC12 cells and investigated as to how their presence influenced cell behaviour and function. Cell morphology was analysed as the QD concentrations were varied over co-incubations up to 72 hours. The QDs were found to be excellent fluorophores, illuminating the cytoplasm of the cells and no deleterious effects were witnessed at concentrations of ~10-9 M. Three assays were utilised to probe how individual cell functions (viability, DNA quantification and proliferation) were affected by the presence of the QDs at various concentrations and incubation times. Cell response was found to not only be concentration dependant but also influenced by the surface environment of the QDs. Gelatine capping on the surface acts as a barrier towards the leaking of toxic atoms, thus reducing the negative impact of the QDs. CONCLUSION: This study has shown that under the correct conditions, QDs can be routinely used for the imaging of PC12 cells with minimal adverse effects. We have found that PC12 cells are highly susceptible to an increased concentration range of the QDs, while the gelatine coating acts as a barrier towards enhanced toxicity at higher QD concentrations.
RESUMEN
Guided neurite growth is critical in both peripheral nervous system and central nervous system nerve regeneration. Scaffolds that provide structural and guidance cues for neuronal cells have a potential role in neural regeneration application. Type I collagen is suitable to be processed as an engineered scaffold for nerve regeneration because of its biological and structural properties. A few previous studies have shown that cross-linking of collagen scaffolds with microbial transglutaminase improves the mechanical strength and degradation properties of the scaffolds. It was shown that laminin 5 can regulate neurite outgrowth and extension. A motif (PPFLMLLKGSTR) in the human laminin 5 alpha 3 chain is crucial for both integrin alpha 3 beta 1 receptor binding and cell adhesion. In the present work, we studied the guidance effect of a laminin peptide (PPFLMLLKGSTR) gradient in collagen and cross-linked collagen scaffolds on neurite growth. Neurites of rat pheochromocytoma (PC12) cells showed a preferential growth toward the high laminin concentration level on the collagen scaffold, while the incorporation of laminin peptide in the scaffold did not influence neurite length of PC12 cells.