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1.
Analyst ; 139(12): 3026-31, 2014 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-24787948

RESUMEN

Lateral flow immunochromatographic rapid diagnostic tests (RDTs) are the primary form of medical diagnostic used for malaria in underdeveloped nations. Unfortunately, many of these tests do not detect asymptomatic malaria carriers. In order for eradication of the disease to be achieved, this problem must be solved. In this study, we demonstrate enhancement in the performance of six RDT brands when a simple sample-processing step is added to the front of the diagnostic process. Greater than a 4-fold RDT signal enhancement was observed as a result of the sample processing step. This lowered the limit of detection for RDT brands to submicroscopic parasitemias. For the best performing RDTs the limits of detection were found to be as low as 3 parasites per µL. Finally, through individual donor samples, the correlations between donor source, WHO panel detection scores and RDT signal intensities were explored.


Asunto(s)
Malaria/diagnóstico , Cromatografía de Afinidad/normas , Humanos , Sensibilidad y Especificidad
2.
Analyst ; 139(7): 1644-52, 2014 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-24503712

RESUMEN

Simple and rapid methods for detecting mRNA biomarkers from patient samples are valuable in settings with limited access to laboratory resources. In this report, we describe the development and evaluation of a self-contained assay to extract and quantify mRNA biomarkers from complex samples using a novel nucleic acid-based molecular sensor called quadruplex priming amplification (QPA). QPA is a simple and robust isothermal nucleic acid amplification method that exploits the stability of the G-quadruplex nucleotide structure to drive spontaneous strand melting from a specific DNA template sequence. Quantification of mRNA was enabled by integrating QPA with a magnetic bead-based extraction method using an mRNA-QPA interface reagent. The assay was found to maintain >90% of the maximum signal over a 4 °C range of operational temperatures (64-68 °C). QPA had a dynamic range spanning four orders of magnitude, with a limit of detection of ~20 pM template molecules using a highly controlled heating and optical system and a limit of detection of ~250 pM using a less optimal water bath and plate reader. These results demonstrate that this integrated approach has potential as a simple and effective mRNA biomarker extraction and detection assay for use in limited resource settings.


Asunto(s)
Técnicas Biosensibles/métodos , Cartilla de ADN/genética , G-Cuádruplex , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Mensajero/análisis , Secuencia de Bases , Técnicas Biosensibles/instrumentación , Dicroismo Circular , Cartilla de ADN/química , Diseño de Equipo , Humanos , Imanes , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Espectrometría de Fluorescencia , Xantopterina/análogos & derivados , Xantopterina/química
3.
Philos Trans A Math Phys Eng Sci ; 362(1818): 1019-36, 2004 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15306482

RESUMEN

Chaotic advection can play an important role in efficient microfluidic mixers. We discuss a design paradigm that exploits chaotic advection and illustrate by two recent examples, namely enhancing gene expression profiling and constructing an in-line microfluidic mixing channel, how application of this paradigm has led to successful micromixers. We suggest that 'designing for chaos', that is, basing practical mixer design on chaotic advection analysis, is a promising approach to adopt in this developing field which otherwise has little to guide it and is constrained by issues of scale and manufacturability.


Asunto(s)
Mezclas Complejas/química , Diseño de Equipo/métodos , Microquímica/métodos , Microfluídica/métodos , Modelos Químicos , Nanotecnología/métodos , Dinámicas no Lineales , Simulación por Computador , Diseño Asistido por Computadora , Microquímica/instrumentación , Microfluídica/instrumentación , Modelos Estadísticos , Movimiento (Física) , Nanotecnología/instrumentación , Soluciones
4.
Ann Biomed Eng ; 29(8): 638-47, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11556720

RESUMEN

Diabetic retinopathy is the leading cause of blindness in working age individuals in the United States. Breakdown of the blood-retinal barrier is one of the earliest events in the progression of diabetic retinopathy. Ideally, therapeutic measures would be directed at this early stage, but there are few sensitive, quantitative methods to assess the retinal vascular barrier in vivo. We present here a method that combines fluorescence microangiography and the simultaneous use of two fluorescent tracers to quantitatively assess the retinal vascular barrier. PS/F (permeability x surface area/flow) describing the retinal vasculature of Long Evans rats was found to be 0.086+/-0.031 (n=13, avg.+/-s.d.). Based on estimates of flow and surface area, we estimate the permeability of sodium fluorescein to be approximately 1.2 x 10(-5) cm/s. Infusion of a hyperosmolar solution of 1.6 M mannitol for 5 min significantly increased PS/F in individual veins and significantly increased a flow weighted PS/F from 0.073+/-0.028 to 0.16+/-0.034 (n=3). In conclusion, we have adapted indicator dilution techniques to quantitatively assess the retinal vasculature in vivo. We have found dual-tracer fluorescence angiography to be a sensitive indicator of increases in the blood-retinal barrier produced by hyperosmolar mannitol. This methodology is a promising new minimally invasive strategy which may be adapted to quantitatively track retinal vascular permeability.


Asunto(s)
Permeabilidad Capilar , Angiografía con Fluoresceína/métodos , Vasos Retinianos/fisiología , Animales , Ingeniería Biomédica , Barrera Hematorretinal , Permeabilidad Capilar/efectos de los fármacos , Retinopatía Diabética/diagnóstico , Retinopatía Diabética/fisiopatología , Fluoresceína , Colorantes Fluorescentes , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Masculino , Manitol/farmacología , Ratas , Vasos Retinianos/efectos de los fármacos , Xantenos
5.
Am J Ophthalmol ; 129(2): 267-9, 2000 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-10682990

RESUMEN

PURPOSE: Transvascular leakage occurs in diabetic retinopathy. The tight junction proteins occludin and zonula occludens-1 (ZO-1) and adherens junction protein cadherin-5 are critical to the maintenance of endothelial barrier. We report a comparison of junction protein expression in the normal and diabetic retina. METHOD: Case report. Postmortem retinal cryosections were prepared from the left eye of a 73-year-old woman with diabetic retinopathy. Cryosections were immunostained for cadherin-5, occludin, and ZO-1 and compared with retinal cryosections from the right eye of a 72-year-old man with no progression of retinal disease. RESULTS: Immunofluorescence showed positive retinal vessel staining for occludin and ZO-1 in both eyes and cadherin-5 in the normal eye but reduced cadherin-5 staining in the retinal vessels of the diabetic eye. CONCLUSION: Increases in transvascular leakage observed in diabetic retinal vasculature may be associated with reduction in the expression of the critical adherens junction protein, cadherin-5.


Asunto(s)
Cadherinas/metabolismo , Retinopatía Diabética/metabolismo , Vasos Retinianos/metabolismo , Anciano , Antígenos CD , Barrera Hematorretinal , Permeabilidad Capilar , Endotelio Vascular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ocludina , Fosfoproteínas/metabolismo , Retina/metabolismo , Proteína de la Zonula Occludens-1
6.
J Biol Chem ; 274(30): 20895-900, 1999 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-10409633

RESUMEN

In this report, we describe the inactivation and site-specific light induction of plasmid expression using a photosensitive caging compound. Plasmids coding for luciferase were caged with 1-(4, 5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) and transfected into approximately 1-cm diameter sites of the skin of rats with particle bombardment. Skin sites transfected with caged plasmids did not express luciferase. However, subsequent exposure of transfected skin sites to 355-nm laser light induced luciferase expression in proportion to the amount of light. Liposome transfection of HeLa cells with DMNPE-caged green fluorescent protein (GFP) plasmids showed similar results. Caging DNA with DMNPE blocks expression at the level of transcription, since in vitro production of mRNA from linearized GFP plasmid was also blocked by caging and subsequently restored by exposure to light. Under the reaction conditions of these experiments, our absorbance data indicate that each DMNPE-caged GFP plasmid contains approximately 270 caging groups. In addition to inhibition and subsequent restoration of plasmid bioactivity, the presence and photocleavage of this relatively small number of cage groups also alters electrophoretic mobility of plasmids and optical absorption characteristics. This light-induced expression strategy provides a new means to target the expression of genetic material with spatial and temporal specificity.


Asunto(s)
ADN/genética , Luciferasas/genética , Nitrobencenos , Transfección/métodos , Animales , Expresión Génica , Marcación de Gen , Genes Reporteros , Células HeLa , Humanos , Luz , Plásmidos/genética , Ratas
7.
Biochim Biophys Acta ; 1445(1): 53-64, 1999 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-10209258

RESUMEN

A critical requirement of gene therapy is expression of the delivered transgene. Transgene expression is facilitated by access to the transcription mechanism found primarily in the nucleus. Factors modulating the interactions between intracellular plasmid and nuclear access are not well understood. In this study, the effect of mitosis on transgene expression was examined by quantitative flow cytometry. Transfection of HeLa cells synchronized at late G1 phase or G2/M phase was performed using a liposomal vector containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dioleoyl-phosphatidylethanolamine (DOPE) (1:1 mol/mol). Cell samples were transfected and subsequently maintained in G1 phase for various durations to modulate the time between plasmid entry and mitosis. The plasmid contains the sequence for a mutated green fluorescent protein (GFP(S65T)) that was used to examine transgene expression. Ethidium monoazide-labeled plasmid was employed to examine the association of plasmid with the cell membrane. The percentage of cells expressing GFP(S65T) increased sharply as the synchronized cell population passed through M phase, suggesting that an event associated with mitosis is essential for transgene expression. Expression levels of the transgene then declined 18 h after mitosis irrespective of transfection strategy. All transfection strategies resulted in the same maximum percentage of GFP(S65T) positive cells (40%) and average GFP(S65T) expression level (3.14x106 molecules per positive cell). Association of plasmid with the cell membrane at late G1 phase was 1.5-fold of that at G2/M phase. These data are evidence for control of transgene expression triggered by events associated with cell cycle.


Asunto(s)
Regulación de la Expresión Génica , Liposomas , Mitosis/genética , Plásmidos/farmacología , Transgenes , Ciclo Celular/genética , Citometría de Flujo/métodos , Terapia Genética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes , Timidina/genética , Timidina/farmacología , Transfección/métodos
8.
Invest Ophthalmol Vis Sci ; 39(12): 2479-85, 1998 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9804158

RESUMEN

PURPOSE: In diabetic retinopathy and macular edema, the blood-retinal barrier fails to function properly, and there is transvascular leakage of proteins and solutes. The tight junction protein occludin and the adherens junction protein cadherin-5 have been shown to be critical to maintaining the endothelial barrier and regulating paracellular transport of large vessel endothelia. However, the expression and distribution of these junction proteins in the retinal endothelium is not well characterized. METHODS: Human and bovine retinal endothelial cells were isolated as described previously. Western blot analysis and flow cytometry techniques were used to assay for the presence of occludin, zonula occludens-1 (ZO-1), cadherin-5, and beta-catenin. The subcellular localization of the proteins was visualized by immunohistochemistry performed on cultured human retinal endothelial cells and cryosections of bovine retina. RESULTS: Western blot analysis and flow cytometry techniques found occludin, ZO-1, cadherin-5, and beta-catenin in cultured human retinal endothelial cells. Immunofluorescence staining of cultured retinal endothelial cells and cryosections of bovine retina showed junctional localization of occludin, ZO-1, cadherin-5, and beta-catenin. CONCLUSIONS: This report demonstrates the expression of occludin and cadherin-5 in retinal endothelial cells and their localization to sites of cell-cell contact. Expression of their respective regulatory proteins, ZO-1 and beta-catenin, at sites of cell-cell contact suggests that occludin and cadherin-5 play a role in maintaining the retinal endothelial barrier.


Asunto(s)
Cadherinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endotelio Vascular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Vasos Retinianos/metabolismo , Uniones Estrechas/fisiología , Transactivadores , Animales , Anticuerpos Monoclonales , Antígenos CD , Barrera Hematorretinal , Western Blotting , Bovinos , Comunicación Celular , Endotelio Vascular/citología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ocludina , Vasos Retinianos/citología , Proteína de la Zonula Occludens-1 , beta Catenina
9.
Invest Ophthalmol Vis Sci ; 39(9): 1676-84, 1998 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9699557

RESUMEN

PURPOSE: To examine the effects of high glucose concentrations on retinal endothelial permeability in an in vitro model of the retinal microvasculature. METHODS: The permeability of the endothelial barrier to small solutes was measured in a chromatographic cell column consisting of bovine retinal endothelial cells cultured on porous fibronectin-coated microcarriers. In each cell column, permeability changes were evaluated by comparing the treatment permeability response over time with the initial baseline permeability. Short-term (2-hour) barrier effects of glucose were examined by measuring permeability at 15-minute intervals after an increase in perfusate concentration from baseline (5.5 mM) to high (25 mM) glucose. Long-term (to 57 days) effects were tested by addition of 25 mM glucose to microcarrier cultures. The effect of glucose on beta-receptor signaling was tested by measuring its effect on the permeability decrease produced by 1 microM isoproterenol. RESULTS: An increase from 5.5 mM to 25 mM glucose concentration did not change retinal endothelial cell monolayer permeability (n=6) during 2 hours. However, an increase in monolayer permeability was observed after 19 days (n=8) in the 25-mM glucose culture. Paralleling this time course, short-term exposure to 25 mM glucose did not prevent a decrease in permeability triggered by the beta-receptor agonist isoproterenol. However, the permeability effect of the agonist was blocked by long-term culture in 25 mM glucose. Permeability of retinal endothelial monolayers cultured in 5.5 mM glucose and treated with 1 microM isoproterenol decreased significantly to 0.71+/-0.06 of baseline (n=4; mean+/-SEM). However, permeability did not change in parallel cell columns made from microcarriers cultured in 25 mM glucose (0.97+/-0.2 of baseline permeability; n=4; mean+/-SEM). CONCLUSIONS: High-glucose culture decreases the retinal endothelial barrier and blocks the response to beta-adrenergic receptors. This model may prove valuable in exploring other hypotheses of increased permeability associated with diabetic retinopathy or other retinal diseases that break down the retinal vascular barrier.


Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Glucosa/farmacología , Receptores Adrenérgicos beta/metabolismo , Vasos Retinianos/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Fluoresceína/metabolismo , Isoproterenol/farmacología , Microscopía Electrónica de Rastreo , Vasos Retinianos/metabolismo , Vasos Retinianos/ultraestructura , Vitamina B 12/metabolismo
10.
Inflammation ; 22(4): 419-33, 1998 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9675612

RESUMEN

We have previously reported that exposure of endothelial monolayers to low (0.12 mM) extracellular calcium significantly decreased the endothelial solute barrier, and that this effect was reversed by restoring 'normal' (1.2 mM) calcium (1). This effect was shown to be dependent on cadherins, however the molecular mechanisms through which barrier was altered by low calcium were not characterized. Here we investigated the mechanism of increased endothelial permeability produced by low calcium exposure. Endothelial permeability was significantly increased by exposure to low (0.12 mM) calcium; this effect was attenuated by pre-treatment with the protein kinase C (PKC) inhibitor, staurosporine (2 x 10(-7) M) for 30 min. Cell border retraction and gap formation produced by low calcium was also prevented by staurosporine. Treatment of monolayers with 0.12 mM calcium also stimulated the endocytosis of endothelial cadherins. This low calcium mediated cadherin endocytosis was also prevented by pretreatment with staurosporine. Low calcium mediated endocytosis was also prevented by the actin filament toxin, cytochalasin D (1 ug/ml, 30 min). We conclude that the mechanism of low calcium mediated loss of endothelial barrier function is mediated in part by a PKC dependent endocytosis of endothelial cadherins, which may involve interactions with the actin cytoskeleton. Physiological regulation of the in vivo endothelial barrier may also involve PKC dependent-actin mediated endocytosis of cadherin junctional elements.


Asunto(s)
Cadherinas/fisiología , Endocitosis/fisiología , Endotelio Vascular/fisiología , Proteína Quinasa C/fisiología , Actinas/fisiología , Animales , Calcio/metabolismo , Calcio/farmacología , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Bovinos , Células Cultivadas , Citocalasina D/farmacología , Citoesqueleto/fisiología , Endocitosis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina/farmacología , Tripsina/metabolismo , Tripsina/farmacología
11.
Am J Physiol ; 274(1): H115-22, 1998 01.
Artículo en Inglés | MEDLINE | ID: mdl-9458859

RESUMEN

We previously reported that platelets release a soluble factor that decreases the solute permeability of cultured bovine aortic endothelial monolayers. This factor was characterized as heat stable, tryspsin sensitive, and not serotonin, adenosine, ADP, or ATP [F. R. Haselton and J. S. Alexander. Am. J. Physiol. 263 (Lung Cell Mol. Physiol. 7): L670-L678, 1992]. We now report its identity as lysophosphatidic acid (LPA). Endothelial permeability decreases rapidly, reversibly, and repeatedly when exposed to platelet supernatants. Continuous exposure produces a sustained decrease in permeability. Methanol extracts of platelet supernatants also decrease endothelial permeability. Treatment of methanol extracts of platelet supernatants with phospholipase B or alkaline phosphatase, which modify the structure of LPA, abolishes the permeability-decreasing activity. However, activity is unaffected by treatment with phospholipase A2. This pattern of enzyme inactivation is consistent with the structure of LPA. Furthermore, synthetic 1-oleoyl-LPA rapidly and significantly decreases endothelial permeability in a concentration-dependent manner. Platelet activation does not appear to be required to produce activity in supernatants from platelet isolations, since P-selectin expression is not increased and thromboxane B2 is < 14 pg/6,000 platelets. Our data show that platelets release a methanol-extractable compound with an enzyme degradation profile consistent with LPA, which decreases the permeability of endothelial monolayers in vitro. In vivo, LPA derived from platelets may be an important mediator of the transport barrier formed by the vascular endothelium.


Asunto(s)
Plaquetas/fisiología , Permeabilidad de la Membrana Celular/fisiología , Endotelio Vascular/fisiología , Lisofosfolípidos/sangre , Fosfatasa Alcalina/metabolismo , Animales , Aorta , Bovinos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Endotelio Vascular/efectos de los fármacos , Feto , Fluoresceína , Humanos , Cinética , Lisofosfolipasa/metabolismo , Lisofosfolípidos/farmacología , Fosfolipasas A/metabolismo , Fosfolipasas A2
12.
J Biol Chem ; 272(41): 25641-7, 1997 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-9325286

RESUMEN

Cationic liposomes are potentially important gene transfer vehicles, although their application has been limited by relatively low efficiency of transgene expression. Single cell quantitative methods, such as those used in this study, should permit a more detailed understanding of the relationships between delivered plasmid and transgene expression. Intracellular plasmid delivery and transgene expression were measured simultaneously using photoconjugated ethidium monoazide as an intracellular plasmid delivery marker and green fluorescent protein (GFP(S65T)) as a transgene expression marker. Quantitative flow cytometry was used to estimate plasmid copy number and GFP(S65T) molecules in single cells. The plasmid was delivered to HeLa cells with a cationic liposome vehicle containing 1,2-dioleoyloxy-3-trimethylammonium-propane and dioleoylphosphatidylethanolamine (1:1 mol/mol). Treatment was carried out continuously for 24 h. Flow cytometry measurements on 20, 000 cells were performed during treatment and for 48 h post-treatment. On a single cell basis, transgene expression efficiency and average GFP(S65T) expression level increased with intracellular plasmid copy number. After 3-h exposure to the liposomal vector, more than 95% of the cells were positive for plasmid entry, but none had detectable transgene expression. Maximum transgene expression was achieved at 24 h and remained unchanged at the 72-h measurement. At 24 h, the average positive cell contained 1.6 x 10(5) plasmid copies and 2.3 x 10(6) GFP(S65T) molecules. Importantly, the measurement strategies revealed that transgene expression varied widely within the entire cell population. Although only 30% of all cells expressed transgene, the subpopulation of cells that rapidly incorporated the vector demonstrated 100% efficiency in transgene expression. This study identifies parameters that modulate highly efficient transgene expression from plasmid delivery by cationic liposomes.


Asunto(s)
Expresión Génica , Liposomas , Plásmidos/genética , Transfección/métodos , Transgenes/genética , Marcadores de Afinidad , Azidas , Cationes , Citometría de Flujo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Cinética , Proteínas Luminiscentes/genética , Plásmidos/metabolismo
13.
J Cell Physiol ; 171(3): 243-51, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9180893

RESUMEN

We investigated the role of the cadherins 5 and 13 in the solute barrier formed by aortic endothelial cells in vitro. In confluent monolayers of bovine aortic endothelial cells, immunofluorescence with antibodies to the external domain of cadherin 5 (Mab 9H7) or to cadherin 13 (Mab Ec6C10) found staining for both cadherins at endothelial cell borders. Western blotting with an antibody to the characteristic cadherin cytoplasmic tail or with an antibody to the extracellular domain of cadherin 5 revealed a single 125 kD protein band. A second larger band was found at 130 kD with the anti-cadherin 13 Mab which was not recognized by an antibody to the cadherin cytoplasmic tail. A calcium switch strategy was used to investigate the involvement of these cadherins in the endothelial barrier. Changes in the permeability of small solutes in an endothelial cell column produced by a decrease in calcium concentration followed by a return to normal calcium, with or without antibody, were recorded. We found that anti-cadherin 5 IgG (10 micrograms/ml) interfered with the reforming of interendothelial junctions after restoration of calcium at every time point tested for a total of 45 min after restoration of calcium. The anti-cadherin 13 IgG (10 micrograms/ml) did not block reforming of the endothelial barrier in a similar manner. The presence of this antibody delayed only by 15 min the restoration of the normal barrier. Without calcium switch, addition of either monoclonal antibody (10 micrograms/ml) to the endothelial cell column had no effect on solute permeability. These results suggest that cadherin 5 in bovine aortic endothelial cells has a major functional role in forming the calcium-sensitive endothelial junction in vitro and may play an important role in the normal structure and function of the in vivo barrier.


Asunto(s)
Cadherinas/metabolismo , Calcio/metabolismo , Endotelio Vascular/metabolismo , Animales , Anticuerpos , Antígenos CD , Western Blotting , Bovinos , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta
14.
Microcirculation ; 3(3): 329-42, 1996 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8930889

RESUMEN

OBJECTIVE: The purpose of this study was to develop an in vitro model which, compared to stagnant two-chamber systems, is more analogous to the rheological conditions found in the vasculature and permits near simultaneous measurement of polymorphonuclear leukocytes (PMN) entrapment and endothelial, monolayer permeability. METHODS: A previously described cell-column model of the vasculature was perfused with 2 x 10(6) PMN/ml at an average fluid velocity of 0.09 cm/s and an average estimated endothelial surface shear stress of 4.5 dyne/cm2 for a total of 60 min. PMN entrapment was estimated from counts of PMN in this recirculating system. Bovine endothelial permeability was estimated from an indicator dilution analysis of the three co-injected dyes: blue dextran (molecular weight [MW]) 2 x 10(6), sodium fluorescein (MW 342) and cyanocobalamin (MW 1355). RESULTS: Circulation of PMN through cell-columns did not activate PMN, although the number of circulating PMN decreased at a rate of 14% per 65 cm2 of endothelial surface in the first 15 min. Endothelial cell monolayer permeability did not change within 60 min. Addition of formyl-methionyl-leucyl-phenylalanine (fMLP) (10(-5) M) activated PMN and significantly increased entrapment of circulating PMN to 27% per 65 cm2 in the first 15 min. Although the percentage of circulating PMN entrapped increased, endothelial monolayer permeability was not increased by either the addition of 10(-5)M fMLP or PMN + fMLP to the cell-column perfusate. CONCLUSIONS: Over the 60-min period studied, entrapment of PMN within a recirculating model of the microvasculature was not associated with an increase in endothelial permeability even after PMN stimulation with fMLP.


Asunto(s)
Endotelio Vascular/citología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Citometría de Flujo , Elastasa de Leucocito/metabolismo , Microcirculación/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Consumo de Oxígeno , Superóxidos/metabolismo , Viscosidad
15.
Exp Eye Res ; 63(2): 211-22, 1996 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8983979

RESUMEN

The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic 'cell-columns'. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3 x 10(4) cells cm-2 of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66 cm in diameter and 1.6 cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2 x 10(6) Da; cyanocobalamin 1355 Da; sodium fluorescein 376 Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57 +/- 0.57 x 10(-5) cm sec-1 for cyanocobalamin and 9.29 +/- 0.78 x 10(-5) cm sec-1 for sodium fluorescein (mean +/- S.E.M., n = 39). Permeability did not increase over 2 hr of cell-column perfusion. Permeability was decreased by 1 micron isoproterenol (n = 3) and increased by 1 microgram ml-1 cytochalasin D (n = 5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.


Asunto(s)
Barrera Hematorretinal/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Animales , Capilares , Bovinos , Células Cultivadas , Citocalasina D/farmacología , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Fluoresceína , Fluoresceínas/farmacocinética , Técnicas de Dilución del Indicador , Isoproterenol/farmacología , Microesferas , Modelos Biológicos , Peso Molecular , Vasos Retinianos/citología , Vasos Retinianos/efectos de los fármacos , Vitamina B 12/farmacocinética
16.
Biotechnol Bioeng ; 50(5): 548-54, 1996 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-18627017

RESUMEN

Cationic liposomes are potentially important gene transfer vehicles, capable of conjugating with anionic DNA by condensation. Flow cytometry was used to examine quantitatively the incorporation of DNA-liposome complex into murine capillary lung endothelial cells. The plasmid DNA, a pSV-beta-galactosidase vector, was covalently labeled with ethidium monoazide by photoactivation. The cationic liposome consisted of egg phosphatidylcholine (90%), cholesterol (5%), and stearylamine (5%). The number of plasmid molecules contained within each cell as a function of exposure time was estimated from fluorescence intensity. Fluorescently labeled plasmid is detectable after 10 min and increases with continued exposure, but at a decreasing rate, up to 2160 min. After 2160 min each cell, on average, contains approximately 10,000 plasmid molecules. Following transfection, a single cell unimodal population was detected by flow cytometry, suggesting that all cells participate in transfection equally. Furthermore, cell cycle analysis indicates that the entry of DNA-liposome complex is independent of cell cycle.

17.
J Appl Physiol (1985) ; 77(5): 2496-505, 1994 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-7868472

RESUMEN

This work examined the relationships between lung microvascular permeability-surface area products (PS) for small solutes in animals of different size and for columns of endothelial-covered microcarrier beads. We assembled PS data (humans, sheep, lambs, and rabbits) for labeled sucrose, mannitol, urea, 1,2-propanediol, 1,3-propanediol, and 1,4-butanediol. In addition, PS for cell columns using sucrose, mannitol, and sodium fluorescein were evaluated. A new mathematical model for the analysis of cell columns that accounts for transit time variations was derived and compared with models neglecting this variation. Allometric relationships between PS and body weight or exchange surface (S) were examined. Permeability relative to diffusivity (P/D) correlated inversely with S for all animals. In addition, P/D for the cell columns fell near this regression line. The results suggest either that permeability for hydrophilic tracers is higher for smaller animals or that the indicator-dilution measurement is a fractal process dependent on scale. Furthermore, the P/D-S correlations may help relate cell column experiments to animal studies.


Asunto(s)
Permeabilidad Capilar/fisiología , Endotelio Vascular/fisiología , Pulmón/irrigación sanguínea , Modelos Biológicos , Animales , Células Cultivadas , Cromatografía , Endotelio Vascular/citología , Femenino , Humanos , Técnicas de Dilución del Indicador , Pulmón/citología , Pulmón/fisiología , Masculino , Matemática , Circulación Pulmonar , Conejos
18.
J Cell Physiol ; 156(3): 610-8, 1993 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8360263

RESUMEN

We investigated the role of cadherins in the solute barrier maintained by endothelial cells in vitro. Cell-column chromatographic measurement of endothelial barrier showed that reducing normal extracellular calcium from 1.2 to 0.12 mM increased endothelial permeability to 250% of baseline after 20 min. Restoring normal calcium restored the barrier within 15 min which remained stable for at least 60 min. We used sulfo-NHS-biotin and anti-cadherin antibodies to characterize endothelial proteins with possible roles in the maintenance of endothelial barrier. The non-specific probe sulfo-NHS-biotin identified at least ten endothelial cell surface proteins, with greatest labelling occurring at molecular weights of 125 and 145 kD. Six proteins, including the 125 and 145 kD proteins, associated with the cytoskeleton. Western blotting for the presence of classical cadherins containing the conserved cytoplasmic sequence CDPTAPPYDSLLVFDYEG detected two bands at 145 and 125 kD which associated with the cytoskeleton. Western blotting with an antibody, which recognizes FHLRAHAVDINGNQV, an extracellular homotypic binding region of N-cadherin, detects three bands. Of these three, one protein had a molecular weight of 125 kD and was associated with the cytoskeleton. Immunofluorescence with both N-cadherin and anti-peptide 1 antibodies found staining at endothelial cell borders. The utility of a newly developed cell-column calcium switch assay was tested by verifying the functional role of the previously described epithelial cadherin, uvomorulin, in epithelial barrier. We then applied this method to endothelial cell columns and found the N-cadherin antibody interfered with the reforming of interendothelial junctions. These results suggest that, as in epithelial cells, cadherins in bovine endothelial cells have a functional role in forming the calcium sensitive endothelial junction and may play an important role in the formation of normal barrier.


Asunto(s)
Cadherinas/fisiología , Permeabilidad Capilar/fisiología , Endotelio Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/fisiología , Avidina , Biotina , Western Blotting , Cadherinas/química , Cadherinas/inmunología , Calcio/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular
19.
J Appl Physiol (1985) ; 74(5): 2493-501, 1993 May.
Artículo en Inglés | MEDLINE | ID: mdl-7687599

RESUMEN

Perilla ketone (PK) is a potent lung toxin that causes increased microvascular permeability pulmonary edema in grazing animals. Because the mechanism of action of PK is not know, we investigated whether PK directly affects endothelial cells. Bovine aortic endothelial cells were grown to confluence on Cytodex-3 microcarrier beads and placed in a chromatographic cell column. Monolayer permeability was evaluated from the elution profiles of three optical tracers: blue dextran (2 x 10(6) mol wt), sodium fluorescein (NaF, 342 mol wt), and cyanocobalamin (B12, 1,355 mol wt). Perfusion with 1.2 mM PK increased permeability within 15 min to NaF and B12 by 51 +/- 6 and 54 +/- 11%, respectively. Permeability returned to baseline after PK removal. These in vitro results suggest that PK produces a rapid and reversible increase in endothelial permeability directly. Staining of fixed cells with rhodamine-phalloidin revealed a major disruption of actin microfilaments after PK treatment. Because previous reports suggested that PK may be activated via cytochrome P-450, we attempted to block this using the cytochrome P-450 inhibitor ketoconazole. Ketoconazole alone did not significantly affect permeability, and the combination of PK and ketoconazole resulted in permeability increases similar to those measured for PK alone. This suggests that PK may not require cytochrome P-450 to increase vascular permeability.


Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Monoterpenos , Terpenos/farmacología , Actinas/metabolismo , Animales , Aorta Torácica/citología , Aorta Torácica/fisiología , Western Blotting , Bovinos , Inhibidores Enzimáticos del Citocromo P-450 , Citoesqueleto/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Cetoconazol/farmacología , Microscopía Fluorescente , Embarazo , Ovinos , Fluoruro de Sodio/metabolismo , Coloración y Etiquetado , Terpenos/antagonistas & inhibidores
20.
J Appl Physiol (1985) ; 74(4): 1581-90, 1993 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-7685754

RESUMEN

The addition of adenosine to perfusates flowing through an in vitro cell-column model of the vasculature decreases the permeability of cell column fetal bovine aortic endothelial monolayers. At 10(-4) M adenosine, cell monolayer permeability to the paracellular tracers polyethylene glycol (mol wt 900) and cyanocobalamin (mol wt 1,355) is significantly decreased within 15 min. In continuous treatments with adenosine for up to 30 min, the permeability reduction is maintained, and removal of adenosine returns permeability to baseline levels within 15 min. The effect of adenosine is concentration dependent, with a 5% reduction in permeability with 10(-6) M adenosine, a 21% reduction with 10(-5) M adenosine, and a 37% reduction with 10(-4) M adenosine. The effects of adenosine are not blocked by the adenosine transport inhibitor dipyridamole. Permeability is significantly reduced by the A2-specific adenosine analogue 5'-(N-cyclopropyl)-carboxamidoadenosine but not by the A1-specific adenosine analogue N6-(L-2-phenylisopropyl)adenosine. In addition, the permeability decrease is blocked by the A2-receptor antagonist 8-phenyltheophylline (10(-5)M). We conclude that adenosine decreases the permeability of bovine fetal aortic endothelial monolayers via endothelial A2-purinoceptors.


Asunto(s)
Adenosina/farmacología , Permeabilidad Capilar/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Adenosina/fisiología , Animales , Permeabilidad Capilar/fisiología , Bovinos , Células Cultivadas , Dextranos , Dipiridamol/farmacología , Endotelio Vascular/fisiología , Polietilenglicoles , Antagonistas Purinérgicos , Receptores Purinérgicos/fisiología , Teofilina/análogos & derivados , Teofilina/farmacología
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