Estudio del riesgo cardiovascular en pacientes con bloqueo del sistema renina-angiotensina a través del proteoma de las vesículas extracelulares circulantes / Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles

Introducción: Las vesículas extracelulares (EV) son liberadas al torrente circulatorio por determinados tipos celulares a consecuencia de procesos de transporte, activación o muerte celular. El recuento de EV circulantes de origen plaquetario y endotelial ha demostrado tener un importante papel como biomarcador de riesgo cardiovascular. Por lo tanto, el proteoma de estas EV podría reflejar los procesos celulares subyacentes en pacientes con hipertensión arterial y albuminuria. Material y métodos: El contenido proteico de las EV circulantes se ha analizado mediante cromatografía líquida acoplada a espectrometría de masas. Las EV han sido aisladas mediante un protocolo de ultracentrifugación optimizado para evitar la contaminación de proteínas del plasma. La pureza de la fracción aislada ha sido verificada mediante microscopia electrónica y confocal, y por citometría de flujo. Resultados: En este estudio mostramos un método de gran rendimiento y pureza para obtener extractos proteicos de EV circulantes de pacientes hipertensos con/sin albuminuria para análisis proteómico. Además, aportamos el proteoma de referencia de EV de estos pacientes, compuesto por 2.463 proteínas, y demostramos que las proteínas transportadas por estas vesículas están relacionadas con procesos cruciales involucrados en el riesgo cardiovascular asociado. Conclusión: El proteoma de las EV circulantes constituye una interesante fuente de indicadores para la evaluación de procesos relacionados con el riesgo cardiovascular en pacientes hipertensos con bloqueo del sistema renina-angiotensina

Introduction: Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. Material and methods: Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. Results: We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. Conclusion: The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage

[Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles]. / Estudio del riesgo cardiovascular en pacientes con bloqueo del sistema renina-angiotensina a través del proteoma de las vesículas extracelulares circulantes.

INTRODUCTION: Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. MATERIAL AND METHODS: Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. RESULTS: We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. CONCLUSION: The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage.

Clinical characteristics of patients sensitized to Siberian Hamster

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Sensitive detection of major food allergens in breast milk: first gateway for allergenic contact during breastfeeding.

Food allergy is recognized as a major public health issue, especially in early childhood. It has been hypothesized that early sensitization to food allergens maybe due to their ingestion as components dissolved in the milk during the breastfeeding, explaining reaction to a food, which has never been taken before. Thus, the aim of this work has been to detect the presence of the food allergens in breast milk by microarray technology. We produced a homemade microarray with antibodies produced against major food allergens. The antibody microarray was incubated with breast milk from 14 women collected from Fundación Jiménez Díaz Hospital. In this way, we demonstrated the presence of major foods allergens in breast milk. The analysis of allergens presented in breast milk could be a useful tool in allergy prevention and could provide us a key data on the role of this feeding in tolerance induction or sensitization in children.

Allergen characterization of Chia Seeds (Salvia hispanica), a new allergenic food

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Molecular characterization of contact urticaria in patients with melon allergy.

BACKGROUND: The relevance of contact allergy to plant-related food has recently emerged. Oral allergy syndrome is one of the most characteristic symptoms of fruit allergy, although it also causes systemic reactions. Plant-food allergy is increasing at the same time as pollen allergy, and fruit-induced allergic contact urticaria could be rising as well. OBJECTIVES: The present study was carried out in order to investigate whether one particular primary melon-peel allergen is responsible for contact urticaria. METHODS: Fourteen patients presenting with contact urticaria after touching melon peel were evaluated. A melon-peel extract was prepared and analysed by immunoblotting using the patients' sera. Molecular characterization of IgE-binding bands was performed using mass spectrometry. Melon-peel lipid transfer protein (LTP) was purified. Inhibition studies and contact challenge with the protein were performed to confirm IgE reactivity to the purified allergen. RESULTS: An IgE-binding band of ~8-9 kDa was observed in an immunoblotting assay with all the patients' sera and was identified as an LTP. The melon-peel LTP was purified in two chromatography steps. Inhibition studies confirmed LTP as a major allergen in patients with melon-peel contact urticaria. Contact challenge with melon-peel LTP was performed in five patients, all of whom had positive results, exhibiting itchy erythema and hives in the area of contact. CONCLUSIONS: This study confirmed our previous findings that melon-peel LTP is a major allergen and is responsible for contact allergy. This knowledge may be used to improve both diagnosis and treatment of patients allergic to melon.

Spanish human proteome project: dissection of chromosome 16.

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.

Two-photon cooperative absorption in colliding cold Na atoms.

Two-photon cooperative absorption is common in solid-state physics. In a sample of trapped cold atoms, this effect may open up new possibilities for the study of nonlinear effects. The experiment described herein starts with two colliding Na atoms in the S hyperfine ground state. The pair absorb two photons, resulting in both a P(1/2) and a P(3/2) atom. This excitation is observed by ionization using an external light source. A simple model that considers only dipole-dipole interactions between the atoms allows us to understand the basic features observed in the experimental results. Both the pair of generated atoms and the photons originating from their decay are correlated and may have interesting applications that remain to be explored.

An odorant-binding protein as a new allergen from Siberian hamster (Phodopus sungorus).

A case of anaphylaxis following a bite from a Siberian hamster (SH; Phodopus sungorus) is described. Skin prick tests with hair, urine and salivary gland extracts from SH were positive, while the tests were negative for hair extracts from other rodents. IgE immunoblotting with the patient serum revealed 3 IgE-binding bands of about 18, 21 and 23 kDa. When the patient's serum was preincubated with rabbit, mouse and gerbil hair extracts, no inhibition of the 3 SH IgE-binding bands was demonstrated. Proteins extracted from the 3 bands were analyzed by N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, and peptides were sequenced. IgE-binding bands were identified as being an odorant-binding protein belonging to the lipocalin family. Analysis of the 3 IgE-binding bands found in the hair, urine and salivary glands of SH showed a new allergenic protein lacking cross-reactivity with allergens from other rodents. The 3 bands likely correspond to isoforms of a single allergen.

Phosphorylation reduces the allergenicity of cow casein in children with selective allergy to goat and sheep milk.

This study aimed to characterize the role of phosphorylation of caseins in selective allergy to goat milk (GM) and sheep milk (SM) in patients with good tolerance to cow milk (CM). We performed skin prick tests with milk and caseins from CM, GM, and SM and immunoblotting and specific immunoglobulin (Ig) E determinations with milk and casein from cow and GM and SM. Sensitization to milk and caseins from goat and sheep was demonstrated in all 3 patients by skin tests, determination of specific IgE, or both. Immunoblotting confirmed that GM/SM proteins but not CM proteins were involved in the allergic symptoms. IgE reacted with several protein bands from the caseins and milk extracts of both sheep and goat. Phosphorylation was involved in the different allergenicity of CM caseins. We report the implication of phosphorylation in the allergenicity of caseins involved in selective allergy to GM and SM.

Identification of sole parvalbumin as a major allergen: study of cross-reactivity between parvalbumins in a Spanish fish-allergic population.

BACKGROUND: Fish allergy is becoming an important health problem in Spain, a country with the third highest level of fish consumption after Japan and Portugal. The most common fish allergens are parvalbumins. In our area, the most widely consumed fish species are lean, such as whiff (Lepidorhombus whiffiagonis) and sole (Solea solea). Adverse reactions to fish are usually related to these species, a fact that is largely unknown to allergists in other countries. OBJECTIVE: The aim of this study was to identify and purify the major allergen implicated in allergic response to sole and evaluate the IgE cross-reactivity of purified parvalbumins from whiff and sole, which are phylogenetically close, and more distant species (i.e. cod and salmon). METHODS: Eighteen Spanish fish-allergic patients with a positive history of type I allergy to fish were recruited from the clinic. Total protein extracts and purified parvalbumins from whiff and sole were tested for their IgE-binding properties by combining two-dimensional Western blotting and mass spectrometry. The extent of cross-reactivity between these parvalbumins along with cod and salmon parvalbumins was investigated by IgE ELISA inhibition assay. RESULTS: An IgE-binding spot of approximately 14 kDa was identified as parvalbumin and confirmed as a major allergen in sole extract, which is recognized by almost 70% of the patients. Whiff parvalbumin was recognized by 83.4% of the patients. High cross-reactivity was determined for all purified parvalbumins by IgE inhibition assay. CONCLUSIONS AND CLINICAL RELEVANCE: Sole and whiff parvalbumin were confirmed as major allergens. The parvalbumins of sole, whiff, cod and salmon were highly cross-reactive, thus suggesting a high amino acid sequence identity between them.

An optimal protocol to analyze the rat spinal cord proteome.

Since the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4-7, 3-11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS) to maximise the benefits of this technology. The value of these techniques is demonstrated here by the identification of several proteins known to be associated with neuroglial structures, neurotransmission, cell survival and nerve growth in the central nervous system. Furthermore this study identified many spinal proteins that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of dysfunction or of recovery after Spinal Cord Injury.