Evaluation of recombinant HP6-Tsag, an 18 kDa Taenia saginata oncospheral adhesion protein, for the diagnosis of cysticercosis.

With the objective of providing inexpensive and reproducible assays for the detection of antibodies indicating exposure to Taenia saginata and Taenia solium, we have evaluated the diagnostic utility of the T. saginata oncosphere adhesion protein (HP6-Tsag), expressed in baculovirus (HP6-Bac) and bacteria (HP6-GST [glutathione S-transferase]), employing enzyme-linked immunosorbent assays (ELISAs) and sera from T. saginata infected cattle, T. solium infected pigs and serum and cerebrospinal fluid (CSF) samples from clinically defined T. solium neurocysticercosis (NCC) patients. The two recombinant proteins were antigenic in all three systems, with the signal to background ratio of the HP6-Bac ELISA slightly higher than that for the HP6-GST ELISA. Assay performance in cattle was similar to previously described peptide-based ELISA assays, although NCC sample sensitivity/specificity was marginally better. The sensitivity of the HP6-Bac and HP6-GST ELISAs was close for active human NCC (77.4 and 80.6% for serum and 76.9 and 73.1% for CSF samples, respectively). In inactive human NCC, however, the sensitivity of the HP6-Bac ELISA was almost twice that of the HP6-GST ELISA. Because peptides are relatively expensive and recombinant proteins are simple and economical to produce, the latter may provide useful reagents for antibody detection in countries with endemic cysticercosis/NCC.

Modulación mediante tratamiento con atorvastatina y/o amlodipino del perfil de proteínas liberadas por placas ateroscleróticas humanas / Modulation of the profile of proteins released by human atherosclerotic plaques through

Introducción. Entre las diversas estrategias seguidas para analizar la compleja estructura de la placa de ateroma, nuestro grupo se ha centrado en el estudio de las proteínas secretadas por la pared vascular, que podrían ser potenciales marcadores plasmáticos. Las estatinas o los agentes bloqueadores de los canales de calcio (BCC) pueden modular los valores de las proteínas circulantes en sujetos con diversas afecciones cardiovasculares. Métodos. La electroforesis bidimensional y la espectrometría de masas (EM) se han aplicado al estudio de proteínas diferencialmente secretadas por placas de ateroma incubadas ex vivo respecto a zonas fibrosas adyacentes. Asimismo, hemos estudiado el efecto modulador de la atorvastatina (10­5 M), el amlodipino (10­6 M) o la combinación de ambos fármacos. Resultados. De una media de 620 proteínas detectadas por gel, se identificaron en total 83 proteínas secretadas por las placas ateromatosas mediante EM: 34 proteínas incrementadas en los sobrenadantes de las placas cultivadas ex vivo respecto a las zonas control y 31 con valores de secreción inferiores en la zona ateromatosa. La adición in vitro de fármacos a las placas de ateroma produjo diferentes efectos en los valores de secreción proteicos, dependiendo del tipo de fármaco y de la proteína considerada, si bien la mayoría de las proteínas identificadas revertía a valores control independientemente del tratamiento. Conclusión. Mediante este análisis proteómico hemos caracterizado nuevas proteínas potencialmente involucradas en la formación e inestabilidad de la placa de ateroma. La modulación por distintos tratamientos farmacológicos de dichos marcadores puede ayudar a comprender nuevos mecanismos de acción de dichos fármacos (AU)

Introduction. Analysis of the complex structure of the atheroma plaque is a classical object of cardiovascular research. We studied proteins secreted by the vascular wall, which could be potential plasma markers. Statins or calcium channel blockers modulate the levels of different circulating proteins in patients with diverse cardiovascular diseases. Methods. Two-dimensional electrophoresis and mass spectrometry were applied to identify and characterize proteins differentially secreted by atheroma plaque samples incubated ex vivo versus adjacent fibrous segments considered as controls. We also analyzed the modulatory effect of atorvastatin (10­5 M), amlodipine (10­6 M) and the combination of both drugs. Results. From an average of 620 proteins detected per gel, a total of 83 proteins secreted by atheroma plaque samples were identified by mass spectrometry: 34 proteins were increased in atheroma plaque supernatants versus control segments while 31 showed decreased secretion levels in atheroma plaques. Different effects were detected after in vitro drug administration to complicated atheroma plaques, depending on the kind of drug and protein considered, although the majority of the proteins identified reverted to normal levels independently of the treatment administered. Conclusion. Proteomic analysis characterized a significant number of novel proteins potentially involved in the formation and instability of complicated atherosclerotic plaques. Modulation of these markers by different drugs may help us to understand new potential mechanisms through which these drugs exert their beneficial effects on atherothrombosis (AU)

Isolation of circulating human monocytes with high purity for proteomic analysis.

We describe a simple method for isolation of human blood monocytes with the high purity (95-98%) required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to paramagnetic beads. The elution conditions of the positive selection step were modified to avoid contamination with albumin. This method is compatible with flow cytometry which was used to assess the purity of the cell population. From 28 mL of blood, 10(7) monocytes with > 96% purity are routinely obtained. From the isolated monocytes 200-250 microg of protein could be recovered. The whole method can be performed in three hours. Similar results were obtained using a negative selection step but with lower purity (92%), increased cost and longer time. After solubilization of monocytes, the proteins were analyzed by two-dimensional gel electrophoresis (2-DE) in the 3-10, 4-7, 6-9 and 6-11 pH range. DNA was the main contaminant that interfered with the 2-DE and it was removed by treatment with DNAse. Image analysis of gels allowed the reproducible detection and quantification of 1500 spots in the 4-7 pH range and more than 2000 spots in total by combining (overlapping) 2-D gels in the 4-7, 6-9 and 6-11 pH range. This method is useful for clinical studies of monocytes from a large number of patients due to its rapidity and reproducibility, which permits comparative analysis of normal versus pathological samples and which allows follow up of the expressed proteins of monocytes from each patient.