Coronary Artery Calcification: Current Concepts and Clinical Implications.

Coronary artery calcification (CAC) accompanies the development of advanced atherosclerosis. Its role in atherosclerosis holds great interest because the presence and burden of coronary calcification provide direct evidence of the presence and extent of coronary artery disease; furthermore, CAC predicts future events independently of concomitant conventional cardiovascular risk factors and to a greater extent than any other noninvasive biomarker of this disease. Nevertheless, the relationship between CAC and the susceptibility of a plaque to provoke a thrombotic event remains incompletely understood. This review summarizes the current understanding and literature on CAC. It outlines the pathophysiology of CAC and reviews laboratory, histopathological, and genetic studies, as well as imaging findings, to characterize different types of calcification and to elucidate their implications. Some patterns of calcification such as microcalcification portend increased risk of rupture and cardiovascular events and may improve prognosis assessment noninvasively. However, contemporary computed tomography cannot assess early microcalcification. Limited spatial resolution and blooming artifacts may hinder estimation of degree of coronary artery stenosis. Technical advances such as photon counting detectors and combination with nuclear approaches (eg, NaF imaging) promise to improve the performance of cardiac computed tomography. These innovations may speed achieving the ultimate goal of providing noninvasively specific and clinically actionable information.

A pilot study of occupational exposure to ultrafine particles during 3D printing in research laboratories.

Introduction: 3D printing is increasingly present in research environments, and could pose health risks to users due to air pollution and particulate emissions. We evaluated the nanoparticulate emissions of two different 3D printers, utilizing either fused filament fabrication with polylactic acid, or stereolithography (SLA) with light curing resin. Methods: Nanoparticulate emissions were evaluated in two different research environments, both by environmental measurements in the laboratory and by personal sampling. Results: The SLA printer had higher nanoparticulate emissions, with an average concentration of 4,091 parts/cm3, versus 2,203 particles/cm3 for the fused filament fabrication printer. The collected particulate matter had variable morphology and elemental composition with a preponderance of carbon, sulfur and oxygen, the main byproducts. Discussion: Our study implies that when considering the health risks of particulate emissions from 3D printing in research laboratories, attention should be given to the materials used and the type of 3D printer.

Cell starvation increases uptake of extracellular Thymosin ß4 and its complexes with calcium.

Cell metastasis is the main cause of cancer mortality. Inhibiting early events during cell metastasis and invasion could significantly improve cancer prognosis, but the initial mechanisms of cell transition and migration are barely known. Calcium regulates cell migration, whilst Thymosin ß4 is a G-actin and iron binding peptide associated with tumor metastasis and ferroptosis. Under normal cell growth conditions, intracellular free calcium ions and Thymosin ß4 concentrations are strictly regulated, and are not influenced by extracellular supplementation. However, cell starvation decreases intracellular Thymosin ß4 and increases extracellular peptide uptake above the normal range. Unexpectedly, cell starvation significantly increases internalization of extracellular Ca2+/Thymosin ß4 complexes. Elucidating the role of Ca2+/Thymosin ß4 in the early events of metastasis will likely be important in the future to develop therapies targeting metastasis.

Spatio-Temporal Changes of Extracellular Matrix (ECM) Stiffness in the Development of the Leech Hirudo verbana.

The invertebrate leech Hirudo verbana represents a powerful experimental animal model for improving the knowledge about the functional interaction between the extracellular matrix (ECM) and cells within the tissue microenvironment (TME), and the key role played by ECM stiffness during development and growth. Indeed, the medicinal leech is characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and in which a rapid spatiotemporal development is well established and easily assessed. Our results show that ECM structural organization, as well as the amount of fibrillar and non-fibrillar collagen are deeply different from hatching leeches to adult ones. In addition, the changes in ECM remodelling occurring during the different leech developmental stages, leads to a gradient of stiffness regulating both the path of migratory cells and their fates. The ability of cells to perceive and respond to changes in ECM composition and mechanics strictly depend on nuclear or cytoplasmic expression of Yes-Associated Protein 1 (YAP1), a key mediator converting mechanical signals into transcriptional outputs, expression, and activation.

Toward the renal vesicle: Ultrastructural investigation of the cap mesenchyme splitting process in the developing kidney.

Background: A complex sequence of morphogenetic events leads to the development of the adult mouse kidney. In the present study, we investigated the morphological events that characterize the early stages of the mesenchymal-to-epithelial transition of cap mesenchymal cells, analyzing in depth the relationship between cap mesenchymal induction and ureteric bud (UB) branching. Design and methods: Normal kidneys of newborn non-obese diabetic (NOD) mice were excised and prepared for light and electron microscopic examination. Results: Nephrogenesis was evident in the outer portion of the renal cortex of all examined samples. This process was mainly due to the interaction of two primordial derivatives, the ureteric bud and the metanephric mesenchyme. Early renal developmental stages were initially characterized by the formation of a continuous layer of condensed mesenchymal cells around the tips of the ureteric buds. These caps of mesenchymal cells affected the epithelial cells of the underlying ureteric bud, possibly inducing their growth and branching. Conclusions: The present study provides morphological evidence of the reciprocal induction between the ureteric bud and the metanephric mesenchyme showing that the ureteric buds convert mesenchyme to epithelium that in turn stimulates the growth and the branching of the ureteric bud.

Dopaminergic inhibition of human neutrophils is exerted through D1-like receptors and affected by bacterial infection.

Dopamine (DA) affects immune functions in healthy subjects (HS) and during disease by acting on D1-like (D1 and D5) and D2-like (D2, D3 and D4) dopaminergic receptors (DR); however, its effects on human polymorphonuclear leukocytes (PMN) are still poorly defined. We investigated DR expression in human PMN and the ability of DA to affect cell migration and reactive oxygen species (ROS) production. Experiments were performed on cells from HS and from patients (Pts) with bacterial infections as well, during the acute phase and after recovery. Some experiments were also performed in mice knockout (KO) for the DRD5 gene. PMN from HS express both D1-like and D2-like DR, and exposure to DA results in inhibition of activation-induced morphological changes, migration and ROS production which depend on the activation of D1-like DR. In agreement with these findings, DA inhibited migration of PMN obtained from wild-type mice, but not from DRD5KO mice. In Pts with bacterial infections, during the febrile phase D1-like DRD5 on PMN were downregulated and DA failed to affect PMN migration. Both D1-like DRD5 expression and DA-induced inhibition of PMN migration were however restored after recovery. Dopaminergic inhibition of human PMN is a novel mechanism which is likely to play a key role in the regulation of innate immunity. Evidence obtained in Pts with bacterial infections provides novel clues for the therapeutic modulation of PMN during infectious disease.

Understanding the Behaviour of Human Cell Types under Simulated Microgravity Conditions: The Case of Erythrocytes.

Erythrocytes are highly specialized cells in human body, and their main function is to ensure the gas exchanges, O2 and CO2, within the body. The exposure to microgravity environment leads to several health risks such as those affecting red blood cells. In this work, we investigated the changes that occur in the structure and function of red blood cells under simulated microgravity, compared to terrestrial conditions, at different time points using biochemical and biophysical techniques. Erythrocytes exposed to simulated microgravity showed morphological changes, a constant increase in reactive oxygen species (ROS), a significant reduction in total antioxidant capacity (TAC), a remarkable and constant decrease in total glutathione (GSH) concentration, and an augmentation in malondialdehyde (MDA) at increasing times. Moreover, experiments were performed to evaluate the lipid profile of erythrocyte membranes which showed an upregulation in the following membrane phosphocholines (PC): PC16:0_16:0, PC 33:5, PC18:2_18:2, PC 15:1_20:4 and SM d42:1. Thus, remarkable changes in erythrocyte cytoskeletal architecture and membrane stiffness due to oxidative damage have been found under microgravity conditions, in addition to factors that contribute to the plasticity of the red blood cells (RBCs) including shape, size, cell viscosity and membrane rigidity. This study represents our first investigation into the effects of microgravity on erythrocytes and will be followed by other experiments towards understanding the behaviour of different human cell types in microgravity.

Medium- and Long-Term Re-Treatment of Root Canals Filled with a Calcium Silicate-Based Sealer: An Experimental Ex Vivo Study.

This study investigated the possibility of re-treating a calcium silicate-based sealer (CSBS), compared to an epoxy-resin sealer (RBS), using rotary instrumentation at different times from obturation (1 month/1 year). Thirty-six human mandibular premolars, extracted as a result of orthodontic or periodontal problems, were instrumented and randomly divided into three groups of 12: BR and BR*, which were filled with CSBS and re-treated after one month and one year of storage, respectively, and AH, which was filled with RBS and re-treated after one month. The same re-treatment protocol was used for all teeth, and the times required for the procedure was recorded. The re-treated specimens were longitudinally sectioned and examined at the stereomicroscope (SM) at 20× magnification. Image J Software was used to process the microphotographs. The percentage of residual filling materials in the root canal and the apical third, the ability to reach working length WL and patency, and the time taken to complete the re-treatment were recorded and analyzed by ANOVA and post hoc Bonferroni test (p = 0.05). Scanning electron microscopy (SEM) and coupled energy-dispersive spectroscopy (EDS) were applied to representative samples to evaluate canal cleanliness and chemical elements. Patency and WL were re-established in all of the teeth. Residual filling materials were retained in all specimens of the three groups. The mean percentage of residual materials was significantly different between BR and BR* (p-value = 0.048), with BR* showing the highest values. The mean time to complete re-treatment was significantly lower for AH, followed by BR (p = 0.0001) and BR* (p = 0.0078). Conclusions: After both medium and long storage periods, the CSBS can be concluded to have been successfully removed from canals with simple anatomy.

Thymosin ß4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis.

Thymosin ß4 (Tß4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tß4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tß4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tß4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tß4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tß4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tß4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tß4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tß4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.

Carotid Artery Plaque Calcifications: Lessons From Histopathology to Diagnostic Imaging.

The role of calcium in atherosclerosis is controversial and the relationship between vascular calcification and plaque vulnerability is not fully understood. Although calcifications are present in ≈50% to 60% of carotid plaques, their association with cerebrovascular ischemic events remains unclear. In this review, we summarize current understanding of carotid plaque calcification. We outline the role of calcium in atherosclerotic carotid disease by analyzing laboratory studies and histopathologic studies, as well as imaging findings to understand clinical implications of carotid artery calcifications. Differences in mechanism of calcium deposition express themselves into a wide range of calcification phenotypes in carotid plaques. Some patterns, such as rim calcification, are suggestive of plaques with inflammatory activity with leakage of the vasa vasourm and intraplaque hemorrhage. Other patterns such as dense, nodular calcifications may confer greater mechanical stability to the plaque and reduce the risk of embolization for a given degree of plaque size and luminal stenosis. Various distributions and patterns of carotid plaque calcification, often influenced by the underlying systemic pathological condition, have a different role in affecting plaque stability. Modern imaging techniques afford multiple approaches to assess geometry, pattern of distribution, size, and composition of carotid artery calcifications. Future investigations with these novel technologies will further improve our understanding of carotid artery calcification and will play an important role in understanding and minimizing stroke risk in patients with carotid plaques.

Undercover Toxic Ménage à Trois of Amylin, Copper (II) and Metformin in Human Embryonic Kidney Cells.

In recent decades, type 2 diabetes complications have been correlated with amylin aggregation, copper homeostasis and metformin side effects. However, each factor was analyzed separately, and only in some rare cases copper/amylin or copper/metformin complexes were considered. We demonstrate for the first time that binary metformin/amylin and tertiary copper (II)/amylin/metformin complexes of high cellular toxicity are formed and lead to the formation of aggregated multi-level lamellar structures on the cell membrane. Considering the increased concentration of amylin, copper (II) and metformin in kidneys of T2DM patients, our findings on the toxicity of amylin and its adducts may be correlated with diabetic nephropathy development.

Living with the enemy: from protein-misfolding pathologies we know, to those we want to know.

Conformational diseases are caused by the aggregation of misfolded proteins. The risk for such pathologies develops years before clinical symptoms appear, and is higher in people with alpha-1 antitrypsin (AAT) polymorphisms. Thousands of people with alpha-1 antitrypsin deficiency (AATD) are underdiagnosed. Enemy-aggregating proteins may reside in these underdiagnosed AATD patients for many years before a pathology for AATD fully develops. In this perspective review, we hypothesize that the AAT protein could exert a new and previously unconsidered biological effect as an endogenous metal ion chelator that plays a significant role in essential metal ion homeostasis. In this respect, AAT polymorphism may cause an imbalance of metal ions, which could be correlated with the aggregation of amylin, tau, amyloid beta, and alpha synuclein proteins in type 2 diabetes mellitus (T2DM), Alzheimer's and Parkinson's diseases, respectively.

Clot characterization by multidisciplinary approach: biochemical and imaging parameters in a hypocoagulative setting. A pilot study.

BACKGROUND: Clot characterization is, to the present days, a multimodal approach: scanning the clot by electron microscopy (SEM) is helpful for the visualization of fibrin structure along with laboratory parameters such as the clot waveform analysis (CWA) and thrombin generation in different settings of clot abnormalities. This study aimed to assess whether the coagulative parameters were consistent with the clot images texture acquired by SEM, and therefore to propose a more generalist and integrative approach to clots classification. DESIGN AND METHODS: In this pilot study, the examined population consists of eight healthy subjects, seven patients affected by Acquired Hemophilia A (AHA) and seven patients treated with Vitamin K Antagonists (VKAs), similar for age and gender. We studied the velocity and acceleration (1st and 2nd derivative of the aPTT) of clot formation (CWA), the thrombin generation, and the clots' scanning by SEM. Images acquired with SEM were then analyzed with the MATLAB software with the "Texture Analysis" methods to perform classification. Among the various texture parameters, we reported Contrast and Energy. RESULTS: Significant differences among healthy subjects, patients with AHA and those treated with VKAs were detected for the coagulative parameters. We found no differences between VKAs and AHA patients. Contrast and energy highlighted a significant difference among the three groups in agreement with the laboratory's parameters. We found no significant differences between VKAs and AHA patients. CONCLUSIONS: The use of SEM, CWA and thrombin generation parameters may be a starting point for studies aimed to demonstrate the general characteristics of clot formation in different clinical conditions with a multiparametric approach.

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs.

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges.

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme.

Multiple Organ Dysfunction Syndrome (MODS) induced by Candida krusei in an Aldabra giant tortoise (Aldabrachelys gigantea) and confirmed by electron microscopy analysis.

A young female Aldabra giant tortoise (Adabrachelys gigantea) was presented with anorexia, ataxia, severe constipation and bloating. Analysis revealed liver disease and collected biopsy diagnosed Candida krusei infection. Despite Itraconazole treatment, the tortoise got worse and died. Full necropsy was performed; microbiology showed Candida krusei presence in liver, but histopathology didn't confirm fungal presence with special stains, so scanning electron microscopy was essential to prove a detailed diagnosis of extensive mycosis.

Study of Endochondral Ossification in Human Fetalcartilage Anlagen of Metacarpals: Comparative Morphology of Mineral Deposition in Cartilage and in the Periosteal Bone Matrix.

The progression of mineral phase deposition in hypertrophic cartilage and periosteal bone matrix was studied in human metacarpals primary ossification centers before vascular invasion began. This study aimed to provide a morphologic/morphometric comparative analysis of the calcification process in cartilage and periosteal osteoid used as models of endochondral ossification. Thin, sequential sections from the same paraffin inclusions of metacarpal anlagen (gestational age between the 20th and 22nd weeks) were examined with light microscopy and scanning electron microscopy, either stained or heat-deproteinated. This process enabled the analysis of corresponding fields using the different methods. From the initial CaPO4 nucleation in cartilage matrix, calcification progressed increasing the size of focal, globular, randomly distributed deposits (size range 0.5-5 µm), followed by aggregation into polycyclic clusters and finally forming a dense, compact mass of calcified cartilage. At the same time, the early osteoid calcification was characterized by a fine granular pattern (size range 0.1-0.5 µm), which was soon compacted in the layer of the first periosteal lamella. Scanning electron microscopy of heat-deproteinated sections revealed a rod-like hydroxyapatite crystallite pattern, with only size differences between the early globular deposits of the two calcifying matrices. The morphology of the early calcium deposits was similar in both cartilage and osteoid, with variations in size and density only. However, integration of the reported data with the actual hypotheses of the mechanisms of Ca concentration suggested that ion transport was linked to the progression of the chondrocyte maturation cycle (with recall of H2 O from the matrix) in cartilage, while ions transport was an active process through the cell membrane in osteoid. Other considered factors were the collagen type specificity and the matrix fibrillar texture. Anat Rec, 301:571-580, 2018. © 2017 Wiley Periodicals, Inc.

Multilayer Microstructure of Idiopathic Epiretinal Macular Membranes.

PURPOSE: To identify the ultramicroscopic structure of idiopathic epiretinal macular membranes (iEMMs) by scanning electron microscopy (SEM). METHODS: We examined 28 iEMMs surgically removed from 28 eyes of 28 patients. All specimens, previously observed at stereomicroscope, were treated with an osmium maceration technique. Fine resolution of iEMMs' 3D architecture and their interaction with the retina were studied using a Philips SEM-FEG XL-30 microscope. RESULTS: The specimens appeared as laminar connective structures partially or completely adherent to the inner limiting membrane (ILM). We identified 4 types of structures: ( 1 ) distinct layers of thin sheets of woven fibers; ( 2 ) folded layers of inhomogeneous thickness of fibrous material more consistent; ( 3 ) thicker and more rigid layers recognizable as collagen fibrils with typical 64-nm period, collagen fibrils isolated or intermingled between them; ( 4 ) lacunar structures with inflammatory and/or necrotic material. The first 3 types of structures appear to thicken towards a centripetal direction from the ILM to the vitreous in order from 1 to 3. The interface of ILM-iEMM tissue shows particular small bridges of connection. Cells are rarely found, especially in the tissue near the ILM. CONCLUSIONS: Layers of various materials follow one another in iEMMs. Cells are rarely found. The interface ILM-iEMM tissue shows particular small bridges of connection. The dynamic modeling of bended layers begins in soft tissue.

Growth and shaping of metacarpal and carpal cartilage anlagen: application of morphometry to the development of short and long bone. A study of human hand anlagen in the fetal period.

A histological and morphometric analysis of human metacarpal and carpal anlagen between the 16th and 22nd embryonic weeks was carried out with the aim of studying the establishment of the respective anlage architecture. No differences in the pattern of growth were documented between the peripheral and central zones of the metacarpal epiphyses and those of the carpals. The regulation of longitudinal growth in long bone anlagen occurred in the transition zone between the epiphysis and the diaphysis (homologous to the metaphyseal growth plate cartilage in more advanced developmental stage of the bone). Comparative zonal analysis was conducted to assess the chondrocyte density, the mean chondrocyte lacunar area, the paired chondrocyte polarity in the orthogonal longitudinal and transverse planes, and the lacunar shape transformation in the metacarpal. In transition from epiphysis to diaphysis chondrocyte density decreased and mean lacunar area increased. No significant differences in the chondrocyte maturation cycle were observed between proximal/distal metacarpal epiphyses and the carpal anlagen. The number of paired chondrocyte oriented along the growth vector was significantly higher in both proximal/distal transition zones between epiphysis and diaphysis. Human metacarpals shared with experimental models (like mice and nonmammal tetrapods) an early common chondrocyte maturation cycle but with a different timing due to the slower embryonic and fetal developmental rate of human anlagen.

Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods.

OBJECTIVE: Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation. METHODS: By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer. Cultured HUVEC were analysed for adhesion molecule expression by means of flow cytometry, while the morphological changes were evaluated by means of SEM. Cell adhesion was evaluated by means of flow cytometry and both OM and SEM. RESULTS: HUVEC expressed under resting conditions the adhesion molecules ICAM-1, VCAM-1 and E-selectin and their expression was upregulated by stimulation with TNF-α (0.1-10ng/ml) as well as with LPS (1µg/ml). SEM analysis showed that stimulation with both stimuli profoundly affect cell morphology. Flow cytometric evaluation of cell adhesion showed that the ability of cells to adhere to HUVEC monolayer was quite different in the two preparations, with the lowest adhesion for FB in all the cell subsets analysed. Finally, isolated granulocytes were able to adhere to HUVEC monolayer more than cells identified in FB or BC and the adhesion was increased during activation of HUVEC with 10ng/ml of TNF-α. CONCLUSION: Our data showed that cell manipulation necessary for the isolation of specific immune cells from whole blood profoundly affect the ability of these cells to adhere to the HUVEC monolayer although their functional properties remain unchanged.