Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools.

BACKGROUND AND OBJECTIVES: Microparticles (MPs) are small phospholipid vesicles of less than 1 microm, shed in blood flow by various cell types. These MPs are involved in several biological processes and diseases. MPs have also been detected in blood products; however, their role in transfused patients is unknown. The purpose of this study was to characterize those MPs in blood bank conditions. MATERIALS AND METHODS: Qualitative and quantitative experiments using flow cytometry or proteomic techniques were performed on MPs derived from erythrocytes concentrates. In order to count MPs, they were either isolated by various centrifugation procedures or counted directly in erythrocyte concentrates. RESULTS: A 20-fold increase after 50 days of storage at 4 degrees C was observed (from 3370 +/- 1180 MPs/microl at day 5 to 64 850 +/- 37 800 MPs/microl at day 50). Proteomic analysis revealed changes of protein expression comparing MPs to erythrocyte membranes. Finally, the expression of Rh blood group antigens was shown on MPs generated during erythrocyte storage. CONCLUSIONS: Our work provides evidence that storage of red blood cell is associated with the generation of MPs characterized by particular proteomic profiles. These results contribute to fundamental knowledge of transfused blood products.

PulseCath iVAC 3LTM hemodynamic performance for simple assisted flow.

The PulseCath iVAC 3L? left ventricular assist device is an option to treat transitory left heart failure or dysfunction post-cardiac surgery. Assisted blood flow should reach up to 3 l/min. In the present in vitro model exact pump flow, depending on various frequencies and afterload was examined. Optimal flow was achieved with inflation/deflation frequencies of about 70-80/min. The maximal flow rate was achieved at about 2.5 l/min with a minimal afterload of 22 mmHg. Handling of the device was easy due to the connection to a standard intra-aortic balloon pump console. With increasing afterload (up to a simulated mean systemic pressure of 66 mmHg) flow rate and cardiac support are in some extent limited.

Red blood cell microparticles: clinical relevance.

Microparticles are small phospholipid vesicles of less than 1 µm released into the blood flow by various types of cells such as endothelial, platelet, white or red blood cells. They are involved in many biological and physiological processes including hemostasis. In addition, an elevated number of microparticles in the blood is observed in various pathological situations. In the context of transfusion, erythrocyte-derived microparticles are found in red blood cell concentrates. Their role is not elucidated, and they are considered as a type of storage lesion. The purpose of this review is to present recent data showing that erythrocyte-derived microparticles most likely play a role in transfusion medicine and could cause transfusion complications.

Coronary vasomotor control in obesity and morbid obesity: contrasting flow responses with endocannabinoids, leptin, and inflammation.

OBJECTIVES: This study sought to investigate abnormalities in coronary circulatory function in 2 different disease entities of obese (OB) and morbidly obese (MOB) individuals and to evaluate whether these would differ in severity with different profiles of endocannabinoids, leptin, and C-reactive protein (CRP) plasma levels. BACKGROUND: There is increasing evidence that altered plasma levels of endocannabinoids, leptin, and CRP may affect coronary circulatory function in OB and MOB. METHODS: Myocardial blood flow (MBF) responses to cold pressor test from rest and during pharmacologically induced hyperemia were measured with N-13 ammonia positron emission tomography/computed tomography. Study participants (n = 111) were divided into 4 groups based on their body mass index (BMI) (kg/m(2)): 1) control group (BMI: 20 to 24.9, n = 30); 2) overweight group (BMI: 25 to 29.9, n = 31), 3) OB group (BMI: 30 to 39.9, n = 25); and 4) MOB group (BMI ≥40, n = 25). RESULTS: The cold pressor test-induced change in endothelium-related MBF response (ΔMBF) progressively declined in overweight and OB groups when compared with the control group [median: 0.19 (interquartile range [IQR] 0.08, 0.27) and 0.11 (0.03, 0.17) vs. 0.27 (0.23, 0.38) ml/g/min; p ≤ 0.01, respectively], whereas it did not differ significantly between OB and MOB groups [median: 0.11 (IQR: 0.03, 0.17) and 0.09 (-0.01, 0.19) ml/g/min; p = 0.93]. Compared with control subjects, hyperemic MBF subjects comparably declined in the overweight, OB, and MOB groups [median: 2.40 (IQR 1.92, 2.63) vs. 1.94 (1.65, 2.30), 2.05 (1.67, 2.38), and 2.14 (1.78, 2.76) ml/g/min; p ≤ 0.05, respectively]. In OB individuals, ΔMBF was inversely correlated with increase in endocannabinoid anandamide (r = -0.45, p = 0.044), but not with leptin (r = -0.02, p = 0.946) or with CRP (r = -0.33, p = 0.168). Conversely, there was a significant and positive correlation among ΔMBF and elevated leptin (r = 0.43, p = 0.031) and CRP (r = 0.55, p = 0.006), respectively, in MOB individuals that was not observed for endocannabinoid anandamide (r = 0.07, p = 0.740). CONCLUSIONS: Contrasting associations of altered coronary endothelial function with increases in endocannabinoid anandamide, leptin, and CRP plasma levels identify and characterize OB and MOB as different disease entities affecting coronary circulatory function.

Structural alterations of the coronary arterial wall are associated with myocardial flow heterogeneity in type 2 diabetes mellitus.

PURPOSE: To determine the relationship between carotid intima-media thickness (IMT), coronary artery calcification (CAC), and myocardial blood flow (MBF) at rest and during vasomotor stress in type 2 diabetes mellitus (DM). METHODS: In 68 individuals, carotid IMT was measured using high-resolution vascular ultrasound, while the presence of CAC was determined with electron beam tomography (EBT). Global and regional MBF was determined in milliliters per gram per minute with (13)N-ammonia and positron emission tomography (PET) at rest, during cold pressor testing (CPT), and during adenosine (ADO) stimulation. RESULTS: There was neither a relationship between carotid IMT and CAC (r = 0.10, p = 0.32) nor between carotid IMT and coronary circulatory function in response to CPT and during ADO (r = -0.18, p = 0.25 and r = 0.10, p = 0.54, respectively). In 33 individuals, EBT detected CAC with a mean Agatston-derived calcium score of 44 +/- 18. There was a significant difference in regional MBFs between territories with and without CAC at rest and during ADO-stimulated hyperemia (0.69 +/- 0.24 vs. 0.74 +/- 0.23 and 1.82 +/- 0.50 vs. 1.95 +/- 0.51 ml/g/min; p < or = 0.05, respectively) and also during CPT in DM but less pronounced (0.81 +/- 0.24 vs. 0.83 +/- 0.23 ml/g/min; p = ns). The increase in CAC was paralleled with a progressive regional decrease in resting as well as in CPT- and ADO-related MBFs (r = -0.36, p < or = 0.014; r = -0.46, p < or = 0.007; and r = -0.33, p < or = 0.041, respectively). CONCLUSIONS: The absence of any correlation between carotid IMT and coronary circulatory function in type 2 DM suggests different features and stages of early atherosclerosis in the peripheral and coronary circulation. PET-measured MBF heterogeneity at rest and during vasomotor stress may reflect downstream fluid dynamic effects of coronary artery disease (CAD)-related early structural alterations of the arterial wall.

Phase-sensitive black-blood coronary vessel wall imaging.

Black-blood MR coronary vessel wall imaging may become a powerful tool for the quantitative and noninvasive assessment of atherosclerosis and positive arterial remodeling. Although dual-inversion recovery is currently the gold standard, optimal lumen-to-vessel wall contrast is sometimes difficult to obtain, and the time window available for imaging is limited due to competing requirements between blood signal nulling time and period of minimal myocardial motion. Further, atherosclerosis is a spatially heterogeneous disease, and imaging at multiple anatomic levels of the coronary circulation is mandatory. However, this requirement of enhanced volumetric coverage comes at the expense of scanning time. Phase-sensitive inversion recovery has shown to be very valuable for enhancing tissue-tissue contrast and for making inversion recovery imaging less sensitive to tissue signal nulling time. This work enables multislice black-blood coronary vessel wall imaging in a single breath hold by extending phase-sensitive inversion recovery to phase-sensitive dual-inversion recovery, by combining it with spiral imaging and yet relaxing constraints related to blood signal nulling time and period of minimal myocardial motion.

Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry.

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

Right coronary artery flow velocity and volume assessment with spiral K-space sampled breathhold velocity-encoded MRI at 3 tesla: accuracy and reproducibility.

PURPOSE: To evaluate accuracy and reproducibility of flow velocity and volume measurements in a phantom and in human coronary arteries using breathhold velocity-encoded (VE) MRI with spiral k-space sampling at 3 Tesla. MATERIALS AND METHODS: Flow velocity assessment was performed using VE MRI with spiral k-space sampling. Accuracy of VE MRI was tested in vitro at five constant flow rates. Reproducibility was investigated in 19 healthy subjects (mean age 25.4 +/- 1.2 years, 11 men) by repeated acquisition in the right coronary artery (RCA). RESULTS: MRI-measured flow rates correlated strongly with volumetric collection (Pearson correlation r = 0.99; P < 0.01). Due to limited sample resolution, VE MRI overestimated the flow rate by 47% on average when nonconstricted region-of-interest segmentation was used. Using constricted region-of-interest segmentation with lumen size equal to ground-truth luminal size, less than 13% error in flow rate was found. In vivo RCA flow velocity assessment was successful in 82% of the applied studies. High interscan, intra- and inter-observer agreement was found for almost all indices describing coronary flow velocity. Reproducibility for repeated acquisitions varied by less than 16% for peak velocity values and by less than 24% for flow volumes. CONCLUSION: 3T breathhold VE MRI with spiral k-space sampling enables accurate and reproducible assessment of RCA flow velocity.

Endothelial dysfunction in systemic lupus erythematosus: evaluation with 13N-ammonia PET.

Systemic lupus erythematosus (SLE) affects multiple organs and systems, severely involving the cardiovascular system. The aim of this study was to evaluate the presence of endothelial dysfunction with N-13-ammonia PET in asymptomatic SLE patients. Methods: We enrolled 16 women with SLE and 16 healthy women. Myocardial blood flow (MBF) was quantified in a 64-slice PET/CT scanner at rest, during a cold pressor test (CPT), and during stress. Endothelium-dependent vasodilation index, %Delta MBF, and myocardial flow reserve (MFR) were calculated. Results: There were 16 women in the SLE group (mean age +/- SD, 31.4 +/- 8.3 y) and 16 women in the healthy control group (31.5 +/- 11.1 y). Mean endothelium-dependent vasodilatation index and %Delta MBF were significantly lower in SLE patients (1.18 +/- 0.55 vs. 1.63 +/- 0.65, P = 0.04, and 18 +/- 55 vs. 63 +/- 65, P = 0.04, respectively). MFR was also lower in the SLE group (2.41 +/- 0.59 vs. 2.73 +/- 0.77, P = 0.20). Conclusion: SLE patients who are free of active disease present abnormal coronary flow and endothelial dysfunction. It is necessary to develop and intensify treatment strategies directed to CAD in SLE patients.

Sweet sixteen for ANLS.

Since its introduction 16 years ago, the astrocyte-neuron lactate shuttle (ANLS) model has profoundly modified our understanding of neuroenergetics by bringing a cellular and molecular resolution. Praised or disputed, the concept has never ceased to attract attention, leading to critical advances and unexpected insights. Here, we summarize recent experimental evidence further supporting the main tenets of the model. Thus, evidence for distinct metabolic phenotypes between neurons (mainly oxidative) and astrocytes (mainly glycolytic) have been provided by genomics and classical metabolic approaches. Moreover, it has become clear that astrocytes act as a syncytium to distribute energy substrates such as lactate to active neurones. Glycogen, the main energy reserve located in astrocytes, is used as a lactate source to sustain glutamatergic neurotransmission and synaptic plasticity. Lactate is also emerging as a neuroprotective agent as well as a key signal to regulate blood flow. Characterization of monocarboxylate transporter regulation indicates a possible involvement in synaptic plasticity and memory. Finally, several modeling studies captured the implications of such findings for many brain functions. The ANLS model now represents a useful, experimentally based framework to better understand the coupling between neuronal activity and energetics as it relates to neuronal plasticity, neurodegeneration, and functional brain imaging.