RESUMEN

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

RESUMEN

Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical-basolateral polarity and cell-cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell-cell contact-lacking cells, ouabain did not produce a significant change; (4) Na+/K+ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and ß-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.

Asunto(s)

Ouabaína , Canales de Potasio con Entrada de Voltaje , Animales , Ouabaína/farmacología , Potasio/metabolismo , Canales de Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Células Epiteliales/metabolismo , Mamíferos/metabolismo

RESUMEN

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

Asunto(s)

Comunicación Celular/efectos de los fármacos , Dinoprostona/farmacología , Células Epiteliales/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Células Epiteliales/metabolismo , Uniones Comunicantes/metabolismo , Células de Riñón Canino Madin Darby , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo

RESUMEN

Cannabidiol (CBD) has been used to treat a variety of cancers and inflammatory conditions with controversial results. In previous work, we have shown that breast cancer MCF-7 cells, selected by their response to inflammatory IL-1ß cytokine, acquire a malignant phenotype (6D cells) through an epithelial-mesenchymal transition (EMT). We evaluated CBD as a potential inhibitor of this transition and inducer of reversion to a non-invasive phenotype. It decreased 6D cell viability, downregulating expression of receptor CB1. The CBD blocked migration and progression of the IL-1ß-induced signaling pathway IL-1ß/IL-1RI/ß-catenin, the driver of EMT. Cannabidiol reestablished the epithelial organization lost by dispersion of the cells and re-localized E-cadherin and ß-catenin at the adherens junctions. It also prevented ß-catenin nuclear translocation and decreased over-expression of genes for ∆Np63α, BIRC3, and ID1 proteins, induced by IL-1ß for acquisition of malignant features. Cannabidiol inhibited the protein kinase B (AKT) activation, a crucial effector in the IL-1ß/IL-1RI/ß-catenin pathway, indicating that at this point there is crosstalk between IL-1ß and CBD signaling which results in phenotype reversion. Our 6D cell system allowed step-by-step analysis of the phenotype transition and better understanding of mechanisms by which CBD blocks and reverts the effects of inflammatory IL-1ß in the EMT.

Asunto(s)

Neoplasias de la Mama/metabolismo , Cannabidiol/farmacología , Citocinas/metabolismo , Interleucina-1beta/metabolismo , Neoplasias de la Mama/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Cicatrización de Heridas , beta Catenina/metabolismo

RESUMEN

BACKGROUND/AIMS: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. METHODS: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, ß-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²âº removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. RESULTS: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, ß-catenin and γ-catenin and displaced ß-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and ß-catenin in the plasma membrane after Ca²âº replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of ß-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase. CONCLUSION: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.

Asunto(s)

Uniones Adherentes/efectos de los fármacos , Ouabaína/farmacología , Uniones Adherentes/metabolismo , Animales , Proteína Tirosina Quinasa CSK , Cadherinas/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , Perros , Células de Riñón Canino Madin Darby , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , beta Catenina/metabolismo , gamma Catenina/metabolismo , Familia-src Quinasas/metabolismo

RESUMEN

Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitive α1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.

RESUMEN

Inflammation and oxidative stress play major roles in endothelial dysfunction, and are key factors in the progression of cardiovascular diseases. The aim of this study was to evaluate in vitro the effect of three subfractions (SFs) from the Cucumis sativus aqueous fraction to reduce inflammatory factors and oxidative stress induced by angiotensin II (Ang II) in human microvascular endothelial cells-1 (HMEC-1) cells. The cells were cultured with different concentrations of Ang II and 0.08 or 10 µg/mL of SF1, SF2, or SF3, or 10 µmol of losartan as a control. IL-6 (Interleukin 6) concentration was quantified. To identify the most effective SF combinations, HMEC-1 cells were cultured as described above in the presence of four combinations of SF1 and SF3. Then, the effects of the most effective combination on the expression of adhesion molecules, the production of reactive oxygen species (ROS), and the bioavailability of nitric oxide (NO) were evaluated. Finally, a mass spectrometry analysis was performed. Both SF1 and SF3 subfractions decreased the induction of IL-6 by Ang II, and C4 (SF1 and SF3, 10 µg/mL each) was the most effective combination to inhibit the production of IL-6. Additionally, C4 prevented the expression of adhesion molecules, reduced the production of ROS, and increased the bioavailability of NO. Glycine, arginine, asparagine, lysine, and aspartic acid were the main components of both subfractions. These results demonstrate that C4 has anti-inflammatory and antioxidant effects.

Asunto(s)

Aminoácidos/farmacología , Angiotensina II/toxicidad , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Cucumis sativus , Células Endoteliales/efectos de los fármacos , Inflamación/prevención & control , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Aminoácidos/aislamiento & purificación , Antiinflamatorios/aislamiento & purificación , Antioxidantes/aislamiento & purificación , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Cucumis sativus/química , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Óxido Nítrico/metabolismo , Fitoterapia , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

Asunto(s)

Endocitosis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ouabaína/farmacología , Proteolisis/efectos de los fármacos , Proteínas de Uniones Estrechas/metabolismo , Animales , Células Cultivadas , Perros , Células de Riñón Canino Madin Darby

RESUMEN

The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na(+) and H(2)O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na(+),K(+)-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.

Asunto(s)

Cilios/metabolismo , Células Epiteliales/metabolismo , Ouabaína/química , Animales , Cadherinas/metabolismo , Adhesión Celular , Comunicación Celular , Línea Celular , Proliferación Celular , Claudinas/metabolismo , Perros , Inmunoprecipitación , Espectroscopía de Resonancia Magnética/métodos , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Esteroides/metabolismo , Uniones Estrechas , Factores de Tiempo

RESUMEN

Epithelial cells treated with high concentrations of ouabain (e.g., 1 microM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K(+) pumping nor disturb the K(+) balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J(DEX)). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J(DEX), and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.

Asunto(s)

Células Epiteliales/efectos de los fármacos , Ouabaína/farmacología , Uniones Estrechas/efectos de los fármacos , Animales , Proteína Tirosina Quinasa CSK , Dextranos/química , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Iones , Modelos Biológicos , Potasio/química , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Familia-src Quinasas

RESUMEN

Permeability alterations of microvascular endothelia may be a factor in the plasma leakage produced by dengue virus infection. Confluent monolayers of the human dermal microvascular endothelial cell line HMEC-1 were utilized as an experimental model to study the cellular responses induced by the virus. Infected monolayers showed increased permeability for [(3)H]mannitol, but no changes were observed for 4-70 kDa dextrans at 48 h post-infection (p.i.), a time at which viral titres reached maximal values and 40 % of the cells expressed viral proteins. A further increase in permeability occurred at 72 h, still without evident cytopathic effects on the monolayer. Coinciding with this, actin was reorganized in the infected cells and the tight junction protein occludin was displaced to the cytoplasm. Increments in the thickness of stress fibres and focal adhesions were observed in uninfected cells neighbouring infected cells. Culture medium from infected monolayers induced permeability changes and thickening of actin-containing structures in control cultures that resembled those observed 48 h p.i. Interleukin (IL) 8 was found in culture medium at concentrations ranging from 20 to 100 pg ml(-1). Neutralizing antibodies against IL8 partially inhibited the changes produced by the culture medium as well as those induced by addition of IL8. Genistein inhibited the effect of the culture medium and the phosphorylation of proteins associated with focal adhesions and indicated the participation of tyrosine kinases. These findings suggest that IL8 production by infected monolayers contributes to the virus-induced effect on the cytoskeleton and tight junctions and thereby modifies transendothelial permeability.

Asunto(s)

Permeabilidad de la Membrana Celular/fisiología , Citoesqueleto/fisiología , Virus del Dengue/fisiología , Dengue/fisiopatología , Endotelio Vascular/virología , Interleucina-8/metabolismo , Microcirculación/virología , Uniones Estrechas/fisiología , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Medios de Cultivo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Endotelio Vascular/inmunología , Endotelio Vascular/fisiología , Humanos , Interleucina-8/farmacología , Fosforilación , Proteínas Recombinantes/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/ultraestructura , Proteínas Virales/análisis

RESUMEN

In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma memebrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of memebrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association ot the actin binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with tran-Golgi-derived vesicle. Modifications of the ring were induced subjecting the cells to external Ca²+ switch), or by treatment with drugs known to depolymerize actin filament (cytochalasin D, CD). The distribution of myosin I and actin, both in intact cells and in cellular fractions, was monitored using heterlogous cross-reacting antibodies and phalloidin. The authors identified an isoform of myosin I of approximately 110-125 KDa, homologus to myosin IB of Acanthamoeba, a fraction of wich colocalized with the peripheral actin ring. The association seems transient as, once the ring retracted as result of Ca²+ depletion, or became disroganized by CD, myosin not longer colocalized with the actin fibers but appeared dispersed in the cytoplasm. Furthermore, a signficant fraction of the total myosin I in the cell was associated to Golgi-derived vesicles which could also associate in vitro with actin filaments. The authors' data support, then, the participation of myosin I, in association with actin filaments, in vesicle translocation to and from the cell membrane as proposed for natural epithelia, and provide a further insigh into the structural organization that maintains epithelial cell polatiry in cultured monolayers

Asunto(s)

Animales , Perros , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Aparato de Golgi/metabolismo , Miosinas/metabolismo

RESUMEN

Se describen los resultados del estudio de Enterovirus como agentes productores de meningoencefalitis viral, desde 1990 hasta 1994. Fueron estudiados en este período 546 muestras de heces fecales, 95 líquidos cefalorraquídeos y 1 058 sueros pareados, obtenidos de 1 388 pacientes diagnosticados clínicamente con esta enfermedad. Las muestras para aislamiento viral se inocularon en 2 sistemas celulares diferentes, el mayor número de aislamiento se encontró en células diploides de fibroblasto humano. Las determinaciones de anticuerpos se realizaron por prueba de neutralización (micrométodo) con 11 antígenos de Enterovirus (Echo 4,6,9,11 y 30 y Coxsackie B1,2,3,4,5 y 6) y en períodos epidémicos con el virus aislado. En los años estudiados se produjeron 2 brotes epidémicos; uno por Coxsackie A9 (1990-1991) y otro por Echo 30 (1994). En los sueros pareados se encontró mayor positividad a los Echo 6 y 11(AU)

Asunto(s)

Meningoencefalitis/etiología , Meningoencefalitis/microbiología , Enterovirus/aislamiento & purificación , Heces/microbiología , Líquido Cefalorraquídeo/microbiología , Sueros Inmunes/análisis

RESUMEN

Se describen los resultados del estudio de Enterovirus como agentes productores de meningoencefalitis viral, desde 1990 hasta 1994. Fueron estudiados en este periodo 546 muestras de heces fecales, 95 liquidos cefalorraquideos y 1 058 sueros pareados, obtenidos de 1 388 pacientes diagnosticados clinicamente con esta enfermedad. Las muestras para aislamiento viral se inocularon en 2 sistemas celulares diferentes, el mayor numero de aislamiento se encontro en celulas diploides de fibroblasto humano. Las determinaciones de anticuerpos se realizaron por prueba de neutralizacion (micrometodo) con 11 antigenos de Enterovirus (Echo 4,6,9,11 y 30 y Coxsackie B1,2,3,4,5 y 6) y en periodos epidemicos con el virus aislado. En los anos estudiados se produjeron 2 brotes epidemicos; uno por Coxsackie A9 (1990-1991) y otro por Echo 30 (1994). En los sueros pareados se encontro mayor positividad a los Echo 6 y 11

Asunto(s)

Enterovirus/aislamiento & purificación , Heces/microbiología , Sueros Inmunes/análisis , Líquido Cefalorraquídeo/microbiología , Meningoencefalitis/etiología , Meningoencefalitis/microbiología

RESUMEN

Se utilizó la sublínea CLA-1 obtenida por el clonaje a partir de la línea celular AP-61 (Aedes pseudocutellaris) para intentar el aislamiento de virus dengue de 10 muestras de suero de pacientes en fase aguda con diagnóstico clínico de dengue. Las muestras fueron inoculadas paralelamente en C6/36 y AP-61 como sistemas controles. De las 10 muestras, 6 fueron positivas en los 3 sistemas, lo que evidencia la sencibilidad y la utilidad para el aislamiento de la sublínea celular CLA-1 (AU)

Asunto(s)

Virus del Dengue/aislamiento & purificación , Línea Celular , Células Clonales

RESUMEN

Se estandarizó un ELISA para la detección de anticuerpos monoclonales a las proteínas E y NS1 del virus dengue. Se aplicó un ELISA indirecto utilizando como fuente de antígeno células C6/36 inoculadas con la cepa A-15 aislada durante la epidemia de dengue 2 en 1981. Estas células fueron fijadas en placas ELISA a una concentración de 200 000 células/pozo. Se utilizó un control celular en condiciones similares. Para normalizar el sistema se emplearon anticuerpos monoclonales específicos a ambas proteínas. Se realizaron estudios a diferentes tiempos de incubación para determinar el momento de mayor expresión de estas proteínas en la membrana celular. Los resultados muestran una respuesta máxima a las 72 horas posinoculación para ambas proteínas, se obtuvo una sensibilidad para la detección de NS1 de 14,7 ng/mL y para la E de 1,43 ng/mL. Este sistema permite el tamizaje primario de anticuerpos monoclonales a las proteínas E y NS1 del virus dengue 2.

RESUMEN

Se estandarizó un ELISA para la detección de anticuerpos monoclonales a las protreínas E y NS1 del virus dengue.Se aplicó un ELISA indirecto utilizando como fuente de antígeno células C6/36 inoculadas con la cepa A-15 aislada durante la epidemia de dengue 2 en 1981. Estas células fueron fijadas en placas ELISA a una concentración de 200000 células/pozo. Se utilizó un control celular en condiciones similares. Para normalizar el sistema se emplearon monoclonales específicos a ambas proteínas. Se realizaron estudios a diferentes tiempos de incubación para determinar el momento de mayor expresión de estas proteínas en la membrana celular. Los resultados muestran una respuesta máxima a las 72 horas posinoculación para ambas proteínas, se obtuvo una sensibilidad para la detección de NS1 de 14,7 ng/mL y para la E de 1,43 ng/mL. Este sistema permite el tamizaje primario de anticuerpos monoclonales a las proteínas E y NS1 del virus dengue 2 (AU)

Asunto(s)

Ensayo de Inmunoadsorción Enzimática/normas , Anticuerpos Monoclonales/aislamiento & purificación , Dengue , Aedes

Asunto(s)

Anticuerpos/inmunología , Infecciones por Enterovirus , Neurología

RESUMEN

En el presente reporte se presentan las condiciones necesarias para el desarrollo de placas en los agentes productores de efecto citopático ligero que fueron aislados de muestras de líquido cefalorraquídeo de pacientes con neuropatía epidémica (AU)

Asunto(s)

Humanos , Neuritis/líquido cefalorraquídeo , Efecto Citopatogénico Viral , Células Vero

RESUMEN

En el presente reporte se describen algunos factores que influyen en el crecimiento del agente productor del efecto citopático (ECP) ligero aislado a partir de líquido cefalorraquídeo de pacientes de neuropatía epidémica. Se demostró que la concentración de NaHCO3 fue decisiva para la aparicióndel ECP. Concentraciones determinadas de MnCL2 permitiron la visualización del ECP e incrementaron ekl rendimiento viral (AU)

Asunto(s)

Humanos , Neuritis/líquido cefalorraquídeo , Efecto Citopatogénico Viral , Células Vero , Cebollas