Decrease in Cellular Nanovesicles Concentration in Blood of Athletes More Than 15 Hours After Marathon.

INTRODUCTION: Cellular nanovesicles (CNVs), that are shed from cells, have been recognized as promising indicators of health status. We analyzed the effect of long-distance running on concentration of CNVs, along with some standard blood parameters, in 27 athletes two days before and >15 hours after physical effort. METHODS: CNVs were isolated by repetitive centrifugation and washing of samples, and assessed by flow cytometry. Cholinesterase (ChE) and glutathione S-transferase (GST) activity were measured spectrophotometrically. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) was measured with immunoturbidimetric determination and lipidogram parameters were measured by enzymatic colorimetric assay. Flow cytometry was used for blood cell count and mean platelet volume (MPV) measurement. RESULTS: More than 15 hours after physical effort a decrease was found in CNVs' concentration in isolates from blood (46%; p<0.05), in ChE activity in whole blood (47%; p<0.001), in plasma (34%; p<0.01), and in erythrocyte suspension (54%; p<0.001), as well as in GST activity in erythrocyte suspension (16%; p<0.01) and in IL-6 concentration in plasma (63%; p<0.05). We found no change in GST activity in plasma and in TNF-α concentration in plasma. Correlations (>0.8; p<0.001) between CNVs' concentration and ChE activity, and GST activity, respectively, in erythrocyte suspension were found. CONCLUSION: We found that >15 hours post-physical effort, CNVs' concentration was below the initial value, concomitant with other measured parameters: ChE and GST activity as well as IL-6 concentration, indicating a favorable effect of physical effort on health status. CNVs' concentration and ChE activity in isolates from peripheral blood proved to have potential as indicators of the response of the human body to inflammation after physical effort. Physical activity should be considered as an important factor in preparation of subjects for blood sampling in procedures focusing on CNV-containing diagnostic and therapeutic compounds.

Applicability of extracellular vesicles in clinical studies.

BACKGROUND: Extracellular vesicles (EVs) are submicron cellular fragments that mediate intercellular communication. EVs have in the last decade attracted major interest as biomarkers or platforms for biomarkers of health and disease. To better understand the reasons why despite great expectations and considerable effort, EV-based methods have not yet been introduced into clinical practice, we present a systematic analysis of published results of clinical studies. MATERIALS AND METHODS: Clinical studies on populations of body fluid samples, published from 2010 to including 2015, applying centrifugation of fluid human samples with centrifuge accelerations up to about 25 000 g and flow cytometry for detection of EVs were analysed with respect to statistical significance (p), statistical power (P), clinical significance (CS), defined as the difference between the means divided by the sum of standard deviations, and size of the populations (Nmin ), defined as the number of samples in the smaller group. RESULTS: Final analysis included 65 publications with 716 comparisons reporting 308 (43%) statistically significant differences (P < 0·05), 242 (34%) had statistical power P > 0·8 and 88 (12%) had clinical importance CS > 1·96. None of comparison with CS > 1·96 included populations in which the smaller group consisted of 50 or more samples. CONCLUSIONS: To fulfil claimed expectations for EV-based methods as promising diagnostic tools, more evidence on EV-based mechanisms of diseases should be gathered. Also, the methods of EV harvesting and assessment should be improved to yield better repeatability and thus allow clinical studies with larger number of samples.

Effect of shear stress in the flow through the sampling needle on concentration of nanovesicles isolated from blood.

During harvesting of nanovesicles (NVs) from blood, blood cells and other particles in blood are exposed to mechanical forces which may cause activation of platelets, changes of membrane properties, cell deformation and shedding of membrane fragments. We report on the effect of shear forces imposed upon blood samples during the harvesting process, on the concentration of membrane nanovesicles in isolates from blood. Mathematical models of blood flow through the needle during sampling with vacuumtubes and with free flow were constructed, starting from the Navier-Stokes formalism. Blood was modeled as a Newtonian fluid. Work of the shear stress was calculated. In experiments, nanovesicles were isolated by repeated centrifugation (up to 17,570×g) and washing, and counted by flow cytometry. It was found that the concentration of nanovesicles in the isolates positively corresponded with the work by the shear forces in the flow of the sample through the needle. We have enhanced the effect of the shear forces by shaking the samples prior to isolation with glass beads. Imaging of isolates by scanning electron microscopy revealed closed globular structures of a similar size and shape as those obtained from unshaken plasma by repetitive centrifugation and washing. Furthermore, the sizes and shapes of NVs obtained by shaking erythrocytes corresponded to those isolated from shaken platelet-rich plasma and from unshaken platelet rich plasma, and not to those induced in erythrocytes by exogenously added amphiphiles. These results are in favor of the hypothesis that a significant pool of nanovesicles in blood isolates is created during their harvesting. The identity, shape, size and composition of NVs in isolates strongly depend on the technology of their harvesting.

Effect of carbon black nanomaterial on biological membranes revealed by shape of human erythrocytes, platelets and phospholipid vesicles.

BACKGROUND: We studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: the effect of CB nanomaterial was assessed by the average number and distribution of erythrocyte shape types (discocytes, echinocytes, stomatocytes) and of vesicles in test suspensions, with respect to control suspensions. Ensembles of representative images were created and analyzed using computer aided image processing and statistical methods. In a population study, blood of 14 healthy human donors was incubated with CB nanomaterial. Blood cell parameters (concentration of different cell types, their volumes and distributions) were assessed. RESULTS: We found that CB nanomaterial formed micrometer-sized agglomerates in citrated and phosphate buffered saline, in diluted blood and in blood plasma. These agglomerates interacted with erythrocyte membranes but did not affect erythrocyte shape locally or globally. CB nanomaterial agglomerates were found to mediate attractive interaction between blood cells and to present seeds for formation of agglomerate - blood cells complexes. Distortion of disc shape of resting platelets due to incubation with CB nanomaterial was not observed. CB nanomaterial induced bursting of GUVs while the shape of the remaining vesicles was on the average more elongated than in control suspension, indicating indirect osmotic effects of CB nanomaterial. CONCLUSIONS: CB nanomaterial interacts with membranes of blood cells but does not have a direct effect on local or global membrane shape in physiological in vitro conditions. Blood cells and GUVs are convenient and ethically acceptable methods for the study of effects of various substances on biological membranes and therefrom derived effects on organisms.