Plasma-Derived Extracellular Vesicles as Potential Biomarkers in Heart Transplant Patient with Chronic Chagas Disease.

Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers.

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.

Effect of immunosuppression in miRNAs from extracellular vesicles of colorectal cancer and their influence on the pre-metastatic niche.

Colorectal cancer (CRC) occurs with more aggressiveness in kidney transplant recipients compared to the general population. Immunosuppressive therapy plays a crucial role in the development of post-transplant malignancy. Concretely, cyclosporine A (CsA) has intrinsic pro-oncologic properties, while several studies report a regression of cancer after the introduction of rapamycin (RAPA). However, their effect on the extracellular vesicle (EV) content from CRC cell lines and their relevance in the pre-metastatic niche have not yet been studied. Here, we investigated the effect of RAPA and CsA in EV-miRNAs from metastatic and non-metastatic CRC cell lines and the role of relevant miRNAs transferred into a pre-metastatic niche model. EV-miRNA profiles showed a significant upregulation of miR-6127, miR-6746-5p, and miR-6787-5p under RAPA treatment compared to CsA and untreated conditions in metastatic cell lines that were not observed in non-metastatic cells. From gene expression analysis of transfected lung fibroblasts, we identified 22 shared downregulated genes mostly represented by the histone family involved in chromatin organization, DNA packaging, and cell cycle. These results suggest that EV-miR-6127, miR-6746-5p and miR-6787-5p could be a potential epigenetic mechanism induced by RAPA therapy in the regulation of the pre-metastatic niche of post-transplant colorectal cancer.

Proteomics study of human cord blood reticulocyte-derived exosomes.

Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially discovered as a cargo-disposal mechanism of obsolete proteins in the maturation of reticulocytes into erythrocytes. In this work, we present the first mass spectrometry-based proteomics of human Rex (HuRex). HuRex were isolated from cultures of human reticulocyte-enriched cord blood using different culture conditions and exosome isolation methods. The newly described proteome consists of 367 proteins, most of them related to exosomes as revealed by gene ontology over-representation analysis and include multiple transporters as well as proteins involved in exosome biogenesis and erythrocytic disorders. Immunoelectron microscopy validated the presence of the transferrin receptor. Moreover, functional assays demonstrated active capture of HuRex by mature dendritic cells. As only seven proteins have been previously associated with HuRex, this resource will facilitate studies on the role of human reticulocyte-derived exosomes in normal and pathological conditions affecting erythropoiesis.

Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice.

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.

Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals.

Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

Cuál es el mejor FEV1 para detectar insuficiencia respiratoria crónica en la EPOC estable / Below what FEV1 Should Arterial Blood be Routinely Taken to Detect Chronic Respiratory Failure in COPD?

Introducción: La gasometría arterial es la medición de elección para el diagnóstico de insuficiencia respiratoria crónica en la enfermedad pulmonar obstructiva crónica (EPOC). Se ha sugerido que el FEV1 se sitúe entre el 30 y el 50% del valor teórico para su indicación, pero estas cifras nunca han sido validadas. Objetivo: Identificar los valores de FEV1 post-broncodilatador (BD) y saturación arterial de oxígeno (SaO2) que proporcionen la mejor sensibilidad, especificidad y coeficientes de probabilidad (CP) para el diagnóstico de insuficiencia respiratoria crónica hipoxémica y/o hipercápnica en la EPOC estable. Métodos: Se incluyeron 150 pacientes (39 con PaO2 < 60mmHg [8 kPa] y 14 de ellos con una PaCO2¡Ý50mmHg[6,7 kPa]). Se seleccionaron los mejores puntos de corte de FEV1 post-BD y SaO2 para predecirla insuficiencia respiratoria crónica empleando los CP y las curvas Receiver Operating Characteristic. Resultados: Un FEV1 post-BD igual al 36% y una SaO2 del 90% fueron los mejores valores predictivos de insuficiencia respiratoria hipoxémica y un FEV1 post-BD igual al 33% para la variante hipercápnica. Un FEV1 ¡Ý45% descartó la insuficiencia respiratoria hipoxémica. Conclusión: Un FEV1 post-BD igual al 36% se erige en el mejor punto de corte para predecir adecuadamentetanto la insuficiencia respiratoria hipoxémica como la hipercápnica en el paciente con EPOC estable. Por su parte, una SaO2 del 90% ofrece el mejor valor para la insuficiencia hipoxémica aislada. Estos valores podrían ser considerados para futuras recomendaciones/guías clínicas de la EPOC(AU)

Introduction: To diagnose and assess chronic respiratory failure in stable chronic obstructive pulmonarydisease (COPD) the measurement of arterial blood gases (ABG) is required. It has been suggested that ABGbe determined for this purpose when FEV1 ranges between 50% and 30% predicted, but these thresholdsare not evidence-based.Objective: To identify the post-bronchodilator (BD) FEV1 and arterial oxygen saturation (SaO2) valuesthat provide the best sensitivity, specificity, and likelihood ratio (LR) for the diagnosis of hypoxaemicand/or hypercapnic chronic respiratory failure in stable COPD.Methods: A total of 150 patients were included (39 with PaO2 < 60mmHg[8 kPa], 14 of them with a PaCO2¡Ý50mmHg[6.7 kPa]). The best post-BD FEV1 and SaO2 cut-off points to predict chronic respiratory failurewere selected using the PC and the Receiver Operating Characteristics (ROC) curves.Results: A post-BD FEV1 equal to 36% and an SaO2 of 90% were the best predictive values for hypoxaemicrespiratory failure and a post-BD FEV1 equal to 33% for the hypercapnic variant. An FEV1 ¡Ý45% ruled outhypoxaemic respiratory failure. Conclusion: A post-BD FEV1 of 36% is the best cut-off point to adequately predict both hypoxaemic andhypercapnic respiratory failure in the patient with stable COPD. For its part, an SaO2 of 90% is the bestvalue for isolated hypoxaemic failure. These values could be considered for future clinical recommendations/guidelines for COPD(AU)

Transcobalamin polymorphism and serum holo-transcobalamin in relation to Alzheimer's disease.

Isoforms of the vitamin B(12) carrier protein transcobalamin (TC) might influence its cellular availability and contribute to the association between disrupted single-carbon metabolism and Alzheimer's disease (AD). We therefore investigated the relationships between the TC 776C>G (Pro259Arg) genetic polymorphism, total serum cobalamin and holo-TC levels, and disease onset in 70 patients with clinically diagnosed AD and 74 healthy elderly controls. TC 776C>G polymorphism was also determined for 94 histopathologically confirmed AD patients and 107 controls. Serum holo-TC levels were significantly higher in TC 776C homozygotes (p = 0.04). Kaplan-Meier survival functions differed between homozygous genotypes (Cox's F-Test F(42, 46) = 2.1; p = 0.008) and between 776C homozygotes and heterozygotes (Cox's F test F(46, 108) = 1.7; p = 0.02). Proportionately fewer TC 776C homozygotes appear to develop AD at any given age, but this will require confirmation in a longitudinal study.

Alzheimer's disease and total plasma aminothiols.

BACKGROUND: Plasma homocysteine is elevated in Alzheimer's disease, but little is known regarding levels of related aminothiols in the disease. We therefore determined total plasma homocysteine, cysteine, and glutathione levels in patients and control subjects and investigated their relationship with cognitive scores. METHODS: We performed a prospective, case-controlled survey based in two UK Psychogeriatric Assessment Centres. Fifty patients with features compatible with DSM-IV criteria for primary degenerative dementia of Alzheimer type were recruited together with 57 cognitively intact age- and gender-matched control subjects. Mini-Mental State and Alzheimer's Disease Assessment Scale-Cognitive Subsection (ADAS-Cog) scores were determined for patients and control subjects. Aminothiols were assayed with an automated high-performance liquid chromatography (HPLC) system. RESULTS: Patients had significantly elevated total plasma homocysteine (p <.001) and cysteine (p <.01), but there were no group differences for total plasma glutathione. Glutathione was, however, a highly significant and independent predictor of cognitive scores in patients (p =.002); lower plasma levels were associated with more severe cognitive impairment. CONCLUSIONS: Total plasma homocysteine and cysteine are elevated in Alzheimer's disease, suggesting intact transsulphuration but defective remethylation of homocysteine in the disease. Total plasma glutathione levels in patients correlate with cognitive scores. Taken together, these observations perhaps reflect the differential effects of Alzheimer's disease-related oxidative stress on the two key pathways of homocysteine metabolism.