Fluorescence single particle tracking for sizing of nanoparticles in undiluted biological fluids

While extremely relevant to many life science fields, such as biomedical diagnostics and drug delivery, studies on the size of nanoparticulate matter dispersed in biofluids are missing due to a lack of suitable methods. Here we report that fluorescence single particle tracking (fSPT) with maximum entropy analysis is the first technique suited for accurate sizing of nanoparticles dispersed in biofluids, such as whole blood. After a thorough validation, the fSPT sizing method was applied to liposomes that have been under investigation for decades as nanocarriers for drugs. The tendency of these liposomes to form aggregates in whole blood was tested in vitro and in vivo. In addition, we have demonstrated that the fSPT sizing technique can be used for identifying and sizing natural cell-derived microparticles directly in plasma. fSPT sizing opens up the possibility to systematically study the size and aggregation of endogenous or exogenous nanoparticles in biofluids

Formation de plasmine à la surface cellulaire (de la vésiculation à l'apoptose)

CAEN-BU Médecine pharmacie (141182102) / SudocSudocFranceF

Des microparticules cellulaires dévoilent leur fonction fibrinolytique et protéolytique

Des microparticules cellulaires dont la taille varie entre 0,1 et 1 μm sont émises par des cellules activées ou en apoptose. Elles portent, associées à leur membrane, des protéines indiquant leur origine cellulaire et de la phosphatidylsérine qui transforme le feuillet membranaire externe en surface d’assemblage des facteurs de la coagulation. Cette propriété procoagulante est accentuée par la présence de facteur tissulaire (FT) sur certaines microparticules. Une nouvelle fonction pro-fibrinolytique et protéolytique est maintenant décrite pour des microparticules d’origine tumorale ou endothéliale portant des métalloprotéinases matricielles et/ou des composants du système d’activation du plasminogène. Ces microparticules concentrent le plasminogène à leur surface où il est transformé en plasmine par l’urokinase (uPA, urokinase plasminogen activator) liée à son récepteur uPAR. La fibrinolyse, la migration cellulaire, l’angiogenèse, la dissémination tumorale, ainsi que l’apoptose induite par le détachement cellulaire pourraient ainsi être modifiées par la présence de ces microparticules chargées en plasmine

: Microparticles in neurosciences

Invited ReviewInternational audienceMicroparticles (MPs) are membrane fragments shed by cells activated by a variety of stimuli including serine proteases, inflammatory cytokines, growth factors, and stress inducers. MPs originating from platelets, leukocytes, endothelial cells, and erythrocytes are found in circulating blood at relative concentrations determined by the pathophysiological context. The procoagulant activity of MPs is their most characterized property as a determinant of thrombosis in various vascular and systemic diseases including myocardial infarction and diabetes. An increase in circulating MPs has also been associated with ischemic cerebrovascular accidents, transient ischemic attacks, multiple sclerosis, and cerebral malaria. Recent data indicate that besides their procoagulant components and identity antigens, MPs bear a number of bioactive effectors that can be disseminated, exchanged, and transferred via MPs cell interactions. Furthermore, as activated parenchymal cells may also shed MPs carrying identity antigens and biomolecules, MPs are now emerging as new messengers/biomarkers from a specific tissue undergoing activation or damage. Thus, detection of MPs of neurovascular origin in biological fluids such as CSF or tears, and even in circulating blood in case of blood-brain barrier leakage, would not only improve our comprehension of neurovascular pathophysiology, but may also constitute a powerful tool as a biomarker in disease prediction, diagnosis, prognosis, and follow-up

Plasmin on adherent cells: from microvesiculation to apoptosis.: Cell activation by plasmin: microvesiculation, apoptosis

International audienceCell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents

Role of plasminogen activation in neuronal organization and survival

International audienceWe characterized the interactions between plasminogen and neurons and investigated the associated effects on extracellular matrix proteolysis, cell morphology, adhesion, signaling and survival. Upon binding of plasminogen to neurons, the plasmin formed by constitutively expressed tissue plasminogen activator (tPA) degrades extracellular matrix proteins, leading to retraction of the neuron monolayer that detaches from the matrix. This sequence of events required both interaction of plasminogen with carboxy-terminal lysine residues and the proteolytic activity of plasmin. Surprisingly, 24 h after plasminogen addition, plasmin-detached neurons survived and remained associated in clusters maintaining focal adhesion kinase phosphorylation contrasting with other adherent cell types fully dissociated by plasmin. However, long-term incubation (72 h) with plasminogen was associated with an increased rate of apoptosis, suggesting that prolonged exposure to plasmin may cause neurotoxicity. Regulation of neuronal organization and survival by plasminogen may be of pathophysiological relevance, as plasminogen is expressed in the brain and/or extravasate during vascular accidents or inflammatory processes

Hippocampal subfield volumetry in mild cognitive impairment, Alzheimer's disease and semantic dementia.

International audienceBACKGROUND: Hippocampal atrophy is a well-known feature of Alzheimer's disease (AD), but sensitivity and specificity of hippocampal volumetry are limited. Neuropathological studies have shown that hippocampal subfields are differentially vulnerable to AD; hippocampal subfield volumetry may thus prove to be more accurate than global hippocampal volumetry to detect AD. METHODS: CA1, subiculum and other subfields were manually delineated from 40 healthy controls, 18 AD, 17 amnestic Mild Cognitive Impairment (aMCI), and 8 semantic dementia (SD) patients using a previously developed high resolution MRI procedure. Non-parametric group comparisons and receiver operating characteristic (ROC) analyses were conducted. Complementary analyses were conducted to evaluate differences of hemispheric asymmetry and anterior-predominance between AD and SD patients and to distinguish aMCI patients with or without β-amyloid deposition as assessed by Florbetapir-TEP. RESULTS: Global hippocampi were atrophied in all three patient groups and volume decreases were maximal in the CA1 subfield (22% loss in aMCI, 27% in both AD and SD; all p < 0.001). In aMCI, CA1 volumetry was more accurate than global hippocampal measurement to distinguish patients from controls (areas under the ROC curve = 0.88 and 0.76, respectively; p = 0.05) and preliminary analyses suggest that it was independent from the presence of β-amyloid deposition. In patients with SD, whereas the degree of CA1 and subiculum atrophy was similar to that found in AD patients, hemispheric and anterior-posterior asymmetry were significantly more marked than in AD with greater involvement of the left and anterior hippocampal subfields. CONCLUSIONS: The findings suggest that CA1 measurement is more sensitive than global hippocampal volumetry to detect structural changes at the pre-dementia stage, although the predominance of CA1 atrophy does not appear to be specific to AD pathophysiological processes

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis.: Fibrinolytic microparticles

International audienceBACKGROUND: We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. DESIGN AND METHODS: Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. RESULTS: Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. CONCLUSIONS: Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium