Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids

Exosomes are cell-secreted nanovesicles (40–200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×105 exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.FICYT; Ministerio de Educación; Ministerio de Economía y Competitividad; Gobierno Regional de Asturias; Gobierno Regional de Madri

Efecto de agentes inductores de la proliferación peroxisomal sobre la respuesta celular a estímulos inflamatorios: regulación de la óxido nítrico sintasa inducible y del factor de transcripción NF-KB

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Mayo 09-05-2003Los agentes inductores de la proliferación peroxisomal, entre los que se encuentran agentes hipolipemiantes y antidiabéticos orales, se caracterizan por su capacidad de activar a una subfamilia de receptores nucleares, los PPAR. Estos fármacos presentan efectos beneficiosos en el tratamiento de los procesos aterogénicos que se han relacionado con sus acciones antiinflamatonas. Se ha descrito que los agonistas de PPAR inhiben la expresión de la forma inducible de la óxido nítrico sintasa (NOSi) en distintos modelos experimentales y se ha propuesto que dicha inhibición es debida, al menos en parte, a la inhibición de la actividad transcripcional de NF-d. Nosotros hemos observado que cierios agonistas de PPAR potencian la expresión de la NOSi en células mesangiales activadas con estimulos inflamatonos, con la participación de mecanismos de regulación transmipcional. El NO producido podría contribuir a los efectos anti-inflamatonos de estos agentes. Además todos los agonisias empleados inhibieron la vía de activación de NF-d por 12.14- distintos mecanismos. En el caso de la 15-desoxid prostaglandina Jz - prostaglandina que se produce en las células tras la activación de la ciclooxigenasa-2 (COX-2)-, hemos descrito la modificación covalente de la subunidad p50 de dicho factor, a través de una adición de Michael a la cistcina en posición 62. Esta modificacióii tieiie lugar eii células intactas, como hemos podido demostrar mediante el empleo de una prostaglandina biotiniiada. Por ello la modificación postraduccional de proteinas por "prostanilación" podría constituir un mecanismo de autoregulación de los procesos inflamatonos.The peroxisome proliferators such as hypolipidernic agents and antidiabetic compounds, are characterized by their ability to bind a family of nuclear receptors, the PPAR (peroxisome proliferator activated receptors). These dmgs have been reported to exert beneficial effects on cardiovascular system related to their anti-inflammatoiy actions. It has been described that PPAR agonists inhibit the inducible nitric oxide synthase (iNOS) expression in severai systems. It has been pmposed that this inhibition is due to their negative effect on the NF-KB transcriphonal activity. In the present study, we have observed that certain agonists of PPAR ampli@ cytokine-elicited iNOS induction in mesangial cells through transcriptional mechanisms while inhibiting NF-KB activity. The NO may contribute to the anti-inflammatoiy effects of these agents. In the case of 1 5 - d e o x y - ~ ~ * ~ ~ ~ - ~ rJo1, ~aa n d i n prostaglandm generated by the cell &er the induction of cyclooxygenase-2 (COX-2), we have showed that it covalently modi@ the p50 subunit of NFKB by a Michael's addition to the cysteine 62. This modification takes place in intact cells as demonsmted by using a biotinylated prostaglandin. This mechanism could contribute to the self-regulation of the inflammatory process

White matter lesions in cronic migraine are not associated with changes in pulsatility index

International Headache Congress (IHC) of the International-Headache-Society (18th. Vancouver, CANADA. 2017

White matter lesions in cronic migraine are not associated with cerebral vasoreactivity

International Headache Congress (IHC) of the International-Headache-Society (18th. Vancouver, CANADA. 2017

Characterization of Plasma-Derived Extracellular Vesicles Isolated by Different Methods: A Comparison Study

Extracellular vesicles (EV) are small membrane structures released by cells that act as potent mediators of intercellular communication. The study of EV biology is important, not only to strengthen our knowledge of their physiological roles, but also to better understand their involvement in several diseases. In the field of biomedicine they have been studied as a novel source of biomarkers and drug delivery vehicles. The most commonly used method for EV enrichment in crude pellet involves serial centrifugation and ultracentrifugation. Recently, different protocols and techniques have been developed to isolate EV that imply less time and greater purification. Here we carry out a comparative analysis of three methods to enrich EV from plasma of healthy controls: ultracentrifugation, ExoQuickTM precipitation solution (System Biosciences), and Total Exosome Isolation kit (Invitrogen). Our results show that commercial precipitation reagents are more efficient and enable higher EV enrichment factors compared with traditional ultracentrifugation, although subsequent imaging analysis is not possible with some of them. We hope that this work will contribute to the current research on isolation techniques to assist the progress of clinical applications with diagnostic or therapeutic objectives

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation