EP2517021A1 - Methods for analyzing components of glycan containing microparticles - Google Patents

Abstract

The invention provides methods for isolating, characterizing, comparing, and using novel components of biological fluids that are characterized on the basis of post-translational modifications of cell surface. In particular, Glycan- containing Microparticles (Glyco-MPs) populations have been characterized on basis of their dimension and the presence of specific glycan epitopes (such as polysialic acid) on their surface, Phospholipids (such as phosphatidylserine) and/or cell type- specific antigens can be also used as additional criteria for isolating, characterizing, comparing, and/or Glyco-MPs populations. The novel methods allow the detection and isolation of Glyco-MPs that can be used for defining biomarkers within biological fluids that are relevant in the diagnosis or monitoring diseases, and/or in the evaluation of the therapeutic efficacy of a medical treatment or a candidate drug.

Description

METHODS FOR ANALYZING COMPONENTS OF GLYCAN CONTAINING MICROPARTICLES

The present invention relates to methods for isolating, characterizing, comparing and using specific biological components in the biological fluids that are characterized on the basis of post-translational modifications of cell surface, and that can be used for the medical management of disorders.

BACKGROUND OF THE INVENTION

Despite huge efforts made by the scientific community, there is still an urgent need in identifying novel biomarkers for a number of chronic conditions including metabolic, cardiovascular and neurodegenerative diseases, in particular by using biological fluids which can be more easily obtained and analyzed in clinical settings. For example, selected plasma proteins are now considered as relevant biomarkers of various clinical conditions and the use of "omics" technologies should lead to the discovery of more as more (non-)protein biomarkers and other complex profiles such as biochemical and metabolic signatures (Service R, 2008; Bain J et al, 2009; Turer A et al , 2009).

Biomarker research activities suffer from a major technical problem related to the presence of extremely abundant proteins within biological fluids that mask the less abundant sub-proteome that can provide reliable biomarkers of medical interest. In the case of blood or cerebrospinal fluids, specific analytical and/or protein- depletion technologies are used to exclude proteins such as albumin, immunoglobulins, or transferrin and try circumventing this problem (Thouvenot E et al , 2008; Apweiler R et al , 2009a). However, major technical improvements are still needed for characterizing the more variable, but minor, fractions of circulating proteome that are difficult to separate such as proteins on cell surface membrane, which are of major interest as potential novel drug targets and biomarkers (Lai Z et al, 2009; Cordwell S and Thingholm T, 2009).

In the field of membrane proteomics within biological fluids, a growing number of preclinical and clinical studies indicate circulating microparticles (MPs) as potential biomarkers and/or additional mediators of protein-based signalling through cell surface receptors or transporter. As extensively reviewed (Hugel B et al. , 2005; Miguet L et al , 2007; Doeuvre L et al, 2009; Burnier L et al, 2009), MPs are vesicles having a size comprised between 1 and 0.1 micrometer that are formed by the plasma membrane and shed from apoptotic or otherwise activated cell types in response to various conditions and stimuli (chemicals, growth factors, shear stress, apoptotic signals, etc.). MPs composition and membrane antigens are dependent from the cellular origin and/or the type of stimulus that triggered their generation.

MPs formation is associated with the loss of membrane asymmetry and the exposure of specific phospholipids, such as phosphatidylserine, on the outer leaflet which, together with MPs surface antigen, are responsible of strong procoagulant activity that MPs normally exhibit. Phospholipid-binding agents, in particular proteins (Stace C and Ktistakis N. 2006; Lemmon M, 2008), are used for the affinity- based isolation of MPs, after being separated from cellular components within the biological fluid by centrifugation.

The MPs variable concentration and composition, release mechanism, and activities in biological fluids have been extensively studied in plasma or urine, as well as in cell culture conditions, but other biological fluids, such as cerebrospinal or synovial fluids, have been indicated as containing vesicles features that are similar to those found for plasma MPs. However, many of the studies have not been pursued in a standardized manner regarding the MPs purification from biological fluids (Piccin A et al, 2007). In fact, other types of vesicular particles, called exosomes, are also released after cell activation and can be co-purified with MPs, but they diverge from MPs in size (in general below 0.1 micrometer), surface antigens (given their intracellular origin), and absence of phosphatidylserine, leading to potential confusions and doubts on the actual relevance of some published data (Horstman L et al , 2007; Thery C et al, 2009).

MPs are present in the blood of healthy individuals (being produced in particular by endothelial cells, platelets and other circulating cells) but their absolute levels as well as the proportion of their different cellular origin may dramatically change under several pathological conditions. MPs appear as being released from a large variety of cell types within biological fluids, consequently shedding cell antigens that can be relevant for various biological functions or disorders that are associated to inflammation and apoptosis. Due to the phospholipids, Tissue Factor, and other molecules present on their surface, MPs are generally believed to have noxious properties, related to their high procoagulant activity and capability of impairing endothelial activities, inhibiting Nitric Oxide production, inducing cytokine release, and activating cell proliferation and many other biological pathways (Morel O et al , 2009; Mause S et al, 2005; Leroyer A et al, 2007; Mezentsev A et al, 2005; Essayagh S et al, 2005).

The genesis and role of MPs derived from platelets, endothelial cells and monocytes, in diseases such as thrombocytopenia, paroxysmal nocturnal haemoglobinuria, sickle cell disease and other chronic inflammatory disorders have been established. The levels of MPs have been shown to be significantly increased and correlated with the development of number of different disorders, including diabetes (Koga H et al, 2005; Sabatier F et al , 2002), HIV infection (Mack M et al, 2000), autoimmune and rheumatic diseases (Beyer C and Pisetsky D, 2010), following heart, stem cell or kidney transplantation (Al-Massarani G et al, 2009; Nomura S et al , 2008; Garcia S et al, 2005), neoangiogenesis and cancer (Mostefai H et al, 2008), sepsis (Soriano A et al. 2005), neurological pathologies (Horstman L et al. , 2007) or cerebral malaria (Combes V et al , 2005). MPs have been also detected in cerebrospinal fluid and plasma, for example after traumatic brain injury (Morel N et al , 2008), as well as in pulmonary edema fluid (Bastarache J et al, 2009), and as a consequence to the exposure of blood to carbon nanotubes (Semberova J et al. 2009) or coronary stents (Inoue T et al , 2006).

Qualitative and quantitative analyses of MPs tried to elucidate if and how the composition and/or the concentration of MPs can be associated to activities of potential therapeutic or diagnostic interest using proteomic technologies, in addition to classical flow cytometry analysis. The composition of MPs-associate proteomes have been studied in endothelial cell-derived MPs (Banfi, C et al, 2005), total plasma MPs (Jin M et al, 2005; Smalley D et al , 2007), total urinary MPs (Smalley D et al, 2008a), platelet MPs (Garcia B et al , 2005; Dean W et al, 2009), MPs in stored red blood cells (Rubin O et al, 2008), and atherosclerotic plaques-derived MPs (Mayr M et al, 2008). Even though it has been suggested that MPs-based proteomic studies can provide novel biomarkers, in particular for atherosclerosis and other vascular diseases (Merrick B, 2008; Smalley D et al, 2008b; Boulanger C et al. 2006; Shantsila E, 2009), MPs have not been clinically validated yet as biomarkers, apart from preliminary studies on cell type-specific MPs populations that are released from endothelial cells in patients at high risk for coronary heart disease (Nozaki T et al , 2009).

An important aspect that has been so far not systematically compared and explored within MPs populations is the level and the importance of post-translationally modified (PTM) proteins on their surface. Altered levels of PTM proteins, even without global protein expression changes, are often linked to cellular responses and disease states. The complex and dynamic nature of the PTM proteome represents a major technical challenge but recent improvements have been made in the separation and the characterization of PTM proteins, leading to identification of more precisely defined post-translational modifications (Mirza S and Olivier M, 2008; Tate E, 2008; Krueger K and Srivastava S, 2006). Phosphorylation, glycosylation, ubiquitination, and prenylation are the most common and characterized categories of post-translational modifications but, by including specific variants, more than 300 modifications have been listed, and large repertoires of PTM proteins have been generated in databases such as dbPTM (Lee H et al , 2009) and HPRD (Keshava Prasad J et al, 2009).

Apart from isolated examples of PTM proteins that have been detected using antigen-specific compounds or by proteomics within MPs populations, there are no evidence in the prior art that post-translational modifications may provide a more comprehensive and precise understanding on relevance of MPs for defining biomarkers to be used in clinical proteomics (Apweiler R et al. , 2009a; Apweiler R et al, 2009b).

SUMMARY OF INVENTION

The present invention provides methods for isolating, characterizing, comparing, and using novel components of biological fluids that are characterized within MPs populations on the basis of post-translational modifications present on cell surface. In particular, Glycan-containing Microparticles (Glyco-MPs) populations have been characterized on the basis of their dimension (comprised between 0.1 and 1 microMeter) and the presence of glycan epitopes (such as polysialic acid) on their surface.

The methods involve the isolation of biological fluids (in particular of human, primate, or rodent origin) and the separation of Glyco-MPs from the acellular fraction of such biological fluids using at least a glycan-binding agent in a solid or a liquid phase. Glyco-MPs can be further isolated, characterized, and/or compared using additional binding agents that are specific for cell surface components, such as phospholipids (and in particular phosphatidyl serine) and/or cell specific antigens.

The Glyco-MPs that are obtained by this method can be used to establish the concentration and/or other molecular components (such as cell-specific antigens, phospholipids, or glycans) that differ between control and test subjects (e.g., normal or at risk of a disease, treated or untreated for a disease) and that can be used as biomarkers within biological fluids for diagnosing or monitoring diseases in a subject, and/or for evaluating the therapeutic efficacy of a medical treatment or a candidate drug. Further objects of the invention, including kits and medical methods for isolating and characterizing Glyco-MPs populations, as provided in the Detailed Description.

DESCRIPTION OF THE FIGURES

Figure 1 - Characterization of features that differentiate Glyco-MPs

(sub)populations in biological fluids.

An exemplary process for the characterization of features that differentiate Glyco-MPs (sub)populations in biological fluids involves the isolation of Control Samples (e.g., from one or more healthy or untreated subjects) and of Test Samples (e.g., from one or more disease-affected or treated subjects). Alternatively, the Control Samples and the Test Samples can be different biological fluids obtained from the same subject (e.g. , blood and cerebrospinal fluid, or blood and urine), for evaluating the presence of Glyco-MPs populations in different physiological locations.

The corresponding Glyco-MPs populations are then isolated on the basis of their size and by means of at least a Gly can-binding agent that binds the surface of Glyco-MPs in experimental conditions that allow maintaining their integrity. Binding agents that recognize other components of Glyco-MPs surface (such as phospholipids and/or cell type-specific antigens) can be also used for isolating and characterizing Glyco-MPs (sub) populations in the preferred format (in solid or liquid phase).

The resulting materials are then compared by any of the known methods for analyzing materials of biological origin that can lead to the determination of features that are associated to a Glyco-MPs subpopulation (or to a MPs subpopulation that lacks the glycan epitope associated to a specific Glyco-MPs subpopulation) in specific subjects, biological fluids, and/or patho-physiological conditions. Then, this signature can be used for evaluating the status of a given subject with (or without) the isolation of Glyco-MPs populations.

Figure 2 - Antigen-independent quantitative analysis of Glyco-MPs populations in animal models

This analysis was performed in two rat models with altered metabolism: streptozotocin-injected and ZDF and ZLC rats.

In the first model, blood from male diabetic ZDF or control ZLC rats (n=6 per group; 20-weeks old) was obtained by retro-orbital puncture. Total plasma MPs were prepared as described in the Material & Methods section of Example 1 and stored at -80°C until use. The Annexin V-positive MPs (Panel A) and the PolySia (2-2B antibody)-positive Glyco-MPs (Panel B) populations were stained with fluorescently labelled Annexin V and anti-PolySia 2-2B antibody, respectively, and then quantified by flow cytometry. Data are expressed as Mean ± SEM.

In the second model, male Sprague Dawley rats (8-weeks old, n=8/group) were daily injected with Streptozotocin (20mpk) for 5 days. At the end of the treatment, rats were anesthesized and blood samples were obtained by retro-orbital puncture after a 6 hour fasting. Total plasma MPs were prepared as described in the Material & Methods section of Example 1 and stored at -80°C until use. The Annexin V-positive MPs (Panel C) and the PolySia(2-2B antibody)-positive Glyco-MPs (Panel D) populations were stained with fluorescently labelled Annexin V and anti-PolySia 2-2B antibody, respectively, and then quantified by flow cytometry. Data are expressed as Mean ± SEM.

Figure 3 - Antigen-specific quantitative analysis of Glyco-MPs populations in animal models

Total plasma MPs were prepared as described in the Material & Methods section of Example 1 and stored at -80°C until use. The different MP populations have been detected and quantified by flow cytometry using, as fluorescently labelled binding agents, Annexin V (AnnV), MAA lectin (MAA), an anti-erythroid cells antibody (TER1 19), and anti-CD41 antibody (CD41) in the indicated combinations (Panel A). The level of Glucose and Insulin were measured in the corresponding mice group. The glucose and insulin levels in the corresponding mice groups are provided (Panels B and C). Data are expressed as Mean ± SEM.

Figure 4 - Quantitative analysis of Glyco-MPs populations in human plasma.

In a first assay, blood samples were collected and Platelet Free Plasma fraction is obtained in six fasted individuals. The Annexin V-positive and the PolySia(2-2B antibody)-positive Glyco-MPs populations were stained in these fractions using fluorescently labelled Annexin V and anti-PolySia 2-2B antibody, respectively, and then quantified by flow cytometry. The concentration of the specific MPs populations in each individual (SI , S2, S3, S4, S5, S6) were used to generate a correlation plot (Panel A).

In a second assay, blood samples were collected and Platelet Free Plasma fraction is obtained from two groups of subjects (Control and Coronarian) as indicated in Example 1. Total Annexin V-positive (AnnV+) and Annexin V-/MAA lectin positive (AnnV+ MAA+) MPs populations were detected and quantified by flow cytometry (Panel B). Data are expressed as Mean ± SEM and as fold induction between the two groups of subjects. Figure 5 - Characterization of features that allow distinguishing Glyco-MPs (sub) populations that have specific cell origins.

An exemplary process for the characterization of feature(s) that allow comparing Glyco-MPs (sub)populations having specific cell origins involves the generation and the isolation of Glyco-MPs from primary cells or cell lines having specific origin (e.g. , pancreatic cells, leukocytes, platelets, neuronal cells, endothelial cells, stem cells). Control and Test cell cultures (e.g., differentiated or undifferentiated, treated or untreated, healthy or in an apoptotic state), can be compared by any of the known methods for characterizing materials of biological origin, as described for Figure 1. If this analysis is further confirmed in relevant cell types, it can lead to the determination of normal (or disease-specific) features that can be identified as being associated to Glyco-MPs (sub) populations, such as cell surface antigens having specific cell origins in a given biological fluid, that can be used for the analysis of biological fluids in different categories of subjects (e.g., being treated or not treated, affected or no affected by a disorder) for determining if any of them can be defined as a biomarker associated to a normal or disorder-specific status.

Figure 6 - Antigen-independent quantitative analysis of Glyco-MPs of Control and Test Glyco-MPs populations from a cell line.

Min6 cell cultures were treated for 24 hours with palmitate at two concentrations (1.0 mM or 1.5 mM) or vehicle only (BSA) in serum-free medium. At the end of the treatment, cell culture medium was collected and total Annexin V- positive MPs (Panel A) and PolySia(2-2B antibody)-positive Glyco-MPs (Panel B) were stained with fluorescently labelled Annexin V and anti-PolySia 2-2B antibody, respectively, and then quantified by flow cytometry. The data on the effect of palmitate on Glyco-MP generation in this cell line are representative of three independent experiments. Similar results were obtained with Min6 cells using staurosporin as apoptotic agent (data not shown).

Figure 7 - Quantitative analysis of Glyco-MPs of Control and Test Glyco-MPs populations from cell lines of different origins.

The indicated cell lines were exposed to an apoptotic agent and the

MPs populations were analyzed in terms of percentage of Annexin V-positive MPs subpopulations that correspond to either 2-2B antibody- or MAA-specific Glyco-MPs subpopulations, as determined by flow cytometry (Panel A).

The analysis has been then performed in more detail for two cell lines (HT29, Panel B; HepG2, Panel C) by comparing the data that have been generated by flow cytometry using a series of fluorescently labelled binding agents either at the level of cell surface (as percentages of all cells) or at the level of the concentration of MPs populations (as Annexin V-positive MPs population and as specific Glyco-MPs subpopulations concentration) in the cell culture supernatant. The comparison is made between control and apoptotic conditions as defined in Example 2. The average fold induction for Annexin V-positive MPs population for HT29 and HepG2 cells was of 4.0 and 12.9, respectively

Figure 8 - Qualitative analysis of Glyco-MPs by 2D-gel electrophoresis.

Min6 cell cultures were treated for 24 hours with ImM palmitate in serum-free medium. At the end of the treatment, culture medium was collected and total MPs were prepared for 2D-gel electrophoresis as described in the Material & Methods section of Example 2. Total proteins (Panel A) and glycoproteins (Panel B) were visualized on the same gel by staining it with SYPRO Ruby and ProQ Emerald 488 staining reagents, respectively. After extensive washing steps, images were captured using a Typhoon 9400 laser scanner in the area at low pi and medium molecular weight. Specific proteins that appears in a specific region of the gel has being modified with a glycan are indicated with a code and an arrow. The analysis of the Glyco-MPs proteome that is detected using from cell culture or other biological fluids can be pursued in more details using the technical approaches described in the literature.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on unexpected findings of the presence and distribution of a specific post-translational modification on the surface of MPs populations. These evidences suggest that MPs populations that are isolated within biological fluids on the basis of at least a post-translational modification (such as phosphorylation, glycosylation, ubiquitination, and prenylation) can be used for obtaining information for the medical management of disorders, in particular for defining and comparing biomarkers present in biological fluids.

In particular, the glycan epitopes that are present on the surface of MPs populations can be used for isolating and characterizing a novel and useful MPs population named as Glyco-MPs. Glyco-MPs populations can provide information about the state of a tissue, a cell population, an organ, or a subject through the analysis of biological fluids where MPs populations are released from cells. Changes in the regulation and processing of glycans have been correlated to a number of abnormal physiologic conditions. However, no evidences have been provided so far on the possibility to integrate the information on glycans (and in general on a specific post- translational modification) on the analysis of MPs populations in order to provide novel and useful insight of MPs features (such as concentration, composition, and/or surface antigens) within biological fluids.

As shown in the Examples below, Glyco-MPs populations do not simply represent MPs subpopulations having a specific cell origin, but are possibly the result of the aggregation of MPs populations having different cell origins, independently (or not) from cell type-specific antigens. The detection of the Glyco- MPs populations may serve as a proxy for detection of a relevant MPs population in a phosphatidylserine and/or cell type-independent manner. In fact phosphatidylserine- based MPs can be usefully combined in methods that allow isolating Glyco-MPs subpopulations that represent a more informative MPs subpopulation for proteomic, immunologic, and/or glycomic analysis. In particular, the isolation, the comparison, and the use of Glyco-MPs populations is intended to provide means for a more sensitive detection and characterization of biological features that can be used as biomarkers for a disorder.

A method for identifying Glycan-containing Microparticles (Glyco- MPs) populations in a biological fluid comprises the following steps:

a) Obtaining a sample of a biological fluid sample from a subject; b) Isolating the acellular fraction of said biological fluid;

c) Separating the Glyco-MPs from the said acellular fraction by means of at least a glycan-binding agent.

In a preferred embodiment, the step (c) of the method involves separating the Glyco-MPs populations from the said acellular fraction by means of an agent that binds a specific epitope (such as an N-linked Glycan, an O-linked Glycan, or PolySia-related epitope) as glycan-binding agent. The step (c) of the method may also involve the use of at least one further binding agent recognizing a phospholipid, a protein, a lipid, or a glycan other than the selected glycan epitope (e.g. , an PolySia- related epitope).

The expression "Glyco-MPs" refers to phospholipid-containing vesicles of cellular origin that have a submicron dimension, in particular comprised between 100 and 1000 nanometers, and that are separated from a biological fluid by means of at least a glycan-binding agent.

The origin of the Glyco-MPs can be established on the basis of the presence of molecules that are known to be associated to a cell type, and in particular those localized on the cell surface. Glyco-MPs populations are originated by cell types that form organs or tissues, at the interface between the biological fluids and tissues (i.e. endothelial cells or the blood-brain barrier) or within the biological fluids itself (e.g. , platelets, erythrocytes). Glyco-MPs populations can be also isolated using biological fluids obtained in cell culture conditions from laboratory cell lines and primary cells of animal origin that are isolated from biopsies or biological fluids.

Flow cytometry is a preferred technology for isolating Glyco-MPs populations (and MPs in general for in vitro/ex vivo studies) according to their size and it can be standardized using size-calibrated fluorescent beads (Robert S et al , 2009; Rukoyatkina N et al , 2009). Flow cytometry analysis of Glyco-MPs populations can be performed by adapting technologies that have developed for the measurement, the preparative sorting into distinct size fractions, and the image processing of artificial nanoparticles and liposomes having similar size (van Gaal E et al , 2010; unding A et al, 2008). Combinations of antibodies, labelling agents, and other technical elements for an improved detection of MPs have been recently summarized (Orozco A and Lewis D, 2010; van der Heyde H et al, 201 1). Glyco- MPs populations can be also isolated and characterized using other methods previously reported for MPs populations, including atomic force microscopy (Yuana Y et al 2010), dynamic light scattering (Lawrie A et al. 2009), scanning electronic microscopy or antibody array (Lai S et al, 2009).

The term "separating" refers to both the physical separation and isolation of Glyco-MPs populations (and of MPs populations in general) from a biological fluid (e.g., by microfiltration or centrifugation), and the separation of Glyco-MPs populations (and of MPs populations in general) that can be performed by technologies, such flow cytometry or microscopy, which provide means for detecting images and other quantifiable signals characterizing MPs within a sample.

The expression "biological fluids" refers to any bodily fluid (and fraction thereof) from, excreted by or secreted by any living cell or organism, including but not limited to blood, cerebrospinal fluid, urine, synovial fluid, bronchoalveolar lavage fluid, aqueous/vitreous humor, amniotic fluid, seminal fluid, saliva, nipple aspirate fluid, pulmonary edema fluid, tears, proximal fluid (the fluid derived from the extracellular milieu of tissues), fluids obtained from an abscess (or any other infection or inflammation site) or a joint (if affected by disease such as rheumatoid arthritis, osteoarthritis or septic arthritis). Databases and other repertoires of the proteomes from biological fluids (Li S et al, 2009; Hu S et al, 2006) can be used to compare with results obtained by studying Glyco-MPs populations. This expression preferably applies also to plasma, cell culture supernatant and any other fluid obtained from a preliminary fractionation, depletion, or any other purification of such biological fluids. For instance, cell culture supernatants have been used for studying MPs populations that are released from liver or mesenchymal stem cells (Chen T et al, 2010; Herrera M et al, 2010).

The term "sample" encompasses both an initial aliquot of the biological fluid as well as the product of any manipulation of the initial source of proteins, including but not limited to partial purification, fractionation, enzyme digestion, or other treatment.

The expression "acellular fraction" refers to a fraction of a biological fluid in which cells are absent, for example following a centrifugation, a separation by flow cytometry, or an affinity-based chromatography or sorting. The methods of the invention involve the isolation of biological fluids from humans, primates, rodents, or any other animal presenting an interest for medical or veterinary research. The biological fluids can be obtained by puncture, involving the removal of a volume of at least 0.01 ml {e.g., in smaller animal) or at least 1 ml (e.g. , in human or primates). It is of major importance to ascertain that the biological fluid is not mixed with cells that results from the rupture of tissues that contains the biological fluid (such as arterial or venous walls in the case of blood) during the puncture, thus excluding any contamination from uhdesired tissues and cell types.

The isolation of the acellular fraction of biological fluids can be performed by eliminating any cellular elements having a size superior to 1000 nanometers, as it is made possible by flow cytometry, microfiltration, or centrifugation. Consistently with what described in the literature (Piccin A et al, 2007), the separation of the Glyco-MPs populations from the acellular fraction of a biological fluid can be performed by applying technologies for isolating cell vesicles having a diameter comprised between 100 and 1000 nm, as well as a composition, that is typical of Glyco-MPs populations. These two criteria can be applied in any order that is appropriate for eliminating undesired biological entities that may be present in the biological fluid (such as cell debris, soluble proteins, exosomes, cells, or virus). The centrifugation of the biological fluids can be performed at a speed comprised between l,500g and 15,000g, at a temperature comprised between 15°C and 37°C, and for a time comprised between 1 minutes and 60 minutes should allow the separation of fraction containing the Glyco-MPs populations (the supernatants) from the cells (forming the pellet). Optionally, a further purification step can be performed to isolate Glyco-MPs (but not exosomes) by centrifuging the supernatant obtained above at a speed comprised between 15,000g and 30,000g, at a temperature comprised between 15°C and 37°C, and for a time comprised between 1 and 60 minutes, in order to obtain a pellet formed by Glyco-MPs populations.

The expression "binding agent" refers to any material that can bind to the desired molecule (that is, a component of Glyco-MPs such as a protein, a protein variant, a phospholipid, a glycan, or a lipid) and consequently allow detecting, labelling, and/or separating the structures containg such molecule (i. e. Glyco-MPs) in a sample (i.e. the acellular fraction of a biological fluid), preferably by interacting with components on the surface of Glyco-MPs. The binding agent for the desired molecule can be a natural or recombinant protein (such as an antibody or a protein that binds a cell surface antigen), a peptide, a lectin, a glycan, a nucleic acid, a lipid, a phospholipid, an inorganic compound, a nanomaterial, a nucleic acid, an aptamer or a low molecular weight ligand.

The binding agent for the desired molecule can be labelled. There are numerous methods by which the label can produce a signal detectable by external means, for example, desirably by visual examination or by electromagnetic radiation, heat, and chemical reagents. The label or other signal producing system component can also be bound to a specific binding partner, another molecule or to a support such as beads, using any method known in the art, such as chemically cross-linking or using the biotin-streptavidin system. The label can directly produce a signal, and therefore, additional components are not required to produce a signal. Numerous organic molecules, for example fluorescers (such as FITC, PE, and any other known to be compatible with flow cytometry-based MPs detection), absorb ultraviolet and visible light. Other labels directly produce a signal, such as radioactive isotopes and dyes. Alternatively, the label may need other components to produce a signal, and the signal producing system would then include all the components required to produce a measurable signal, which may include substrates, coenzymes, metal ions, or substances that react with enzymatic products.

The binding agent for the desired molecule (that is, a component of Glyco-MPs surface) can be provided in a liquid phase or in a solid phase (for example, by the immobilization on a bead or a plate from which it can be or not separated) forming thus a complex with the Glyco-MPs once that the acellular fraction of a biological fluid is contacted with such agent. Subsequently, depending on the further uses, such complex can be dissociated (for instance, by temperature or chemical- induced denaturation) or the binding agent for the desired molecule can be kept associated. The term "Glycan" refers to chemical groups also named as sugars or carbohydrates. Glycans can be monomers or polymers of sugar residues, but typically contain at least three sugars, and can be linear or branched. A glycan may include natural sugar residues (e.g., glucose, N- acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, sialic acids) and/or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose).

Glycans can be distinguished in N-linked, O-linked, or C-linked glycans on the basis of the linkage to a glycoconjugate via nitrogen, oxygen, or carbon linkage. The glycoconjugate can be also in the form of an advanced glycation end products, resulting from the non-enzymatic modification of glycans. The relevance of protein glycation has been demonstrated in several pathological conditions, including chronic complications associated to diabetes mellitus and renal failure as well as degenerative changes, and more sensitive and selective methods are now available for identification and quantification of such glycated proteins (Priego Capote F and Sanchez J, 2009; Thornalley P, 2005,).

In the present disclosure, the term "glycan" refers in general to the carbohydrate portion of a glycoconjugate, includes, but is not limited to, glycoproteins, glycolipids, proteoglycans and glycophosphosphingolipids or any other known glycoconjugate that present a glycan epitope. Glycoconjugates are found predominantly on the outer cell wall and in secreted fluids usually consist of O- or N- glycosidic linkages of oligosaccharides (a polymer containing a small number, typically three to ten saccharides) to compatible amino acid side chains in proteins or to lipid moieties. Some glycans also have modifications such as acetylation and sulfation. Glycoconjugates contain cell-surface glycans that have been shown to be important in cell-cell interactions due to the presence on the cell surface of various glycan binding receptors, in addition to the glycoconjugates themselves.

The expression "cell surface glycan" refers to a glycan that is present on the exterior surface of a cell that is, in general, covalently linked to a polypeptide (as part of a cell-surface glycoprotein) or a lipid (as part of a cell membrane glycolipid) and that can be exposed as well on the surface of Glyco-MPs populations. A cell surface glycan are formed by homo- and/or heteropolymers of sugar residues which form specific glycan epitopes (also called giycoepitopes) that are specifically recognized by glycan-binding agent.

The expression "glycan-binding agent" refers to binding agents that specifically bind a glycoepitope. Recognition systems of giycoepitopes include, but is not limited to, antibodies, lectins (of animal, plant, or pathogen origin), enzymes containing carbohydrate recognition domain (CRD), antibodies against glycans, cytokines, chaperone and transport proteins, microbial carbohydrate-binding proteins, glycosaminoglycan-binding proteins, or any other known recognition system for glycan epitopes, without limitation.

Several properties of the cell surface glycans present on Glyco-MPs surface can be determined, with or without using glycan-binding agents, including the mass of part or all of the saccharide structure, the charges of the chemical units of the saccharide, identities of the chemical units of the saccharide, total charge of the saccharide, or total number of sulfates or acetates. The methods that are applicable for identifying such properties include, but are not limited to, capillary electrophoresis, NMR, mass spectrometry (both MALDI and ESI), and HPLC with fluorescence detection. Glycans can be detected and/or analyzed on Glyco-MPs, either in the absence or in presence of proteases or glycosidase. In particular, the structural and functional diversity within glycan epitopes that can be used for isolating and characterized within Glyco-MPs populations may be explored using technologies for comparative glycan analysis (Krishnamoorthy L and Mahal L, 2009) and synthesis (Bernades G et al, 2009), glycan arrays (Taylor M and Drickamer K, 2009), means for interfering protein-glycan interactions (Rek A et al, 2009), bioinformatics methods (Mahal L, 2008), and chemical tools for binding and/or modifying glycans, for example for studying the biology of carbohydrates in neuronal cells (Murrey H and Hsieh- Wilson L, 2008; Yanagisawa M and Yu R, 2007; Hwang H et al, 2010).

Extensive lists of glycans and of the corresponding glycan-binding agents (such as monoclonal antibodies, lectins of plant or animal origin, and chemicals) and related technologies have been published, indicating the specificity of the interaction, the compatibility with the integrity and viability of cells, and their potential use for identifying specific cell types and/or antigens of medical interest (see e.g. , Varki A et al , 2009).

A general overview of glycan epitopes of potential interest and of the corresponding glycan binding agents is provided in Table 1 below. Table 1

Among the glycan-binding agents, lectins are of particular interest given their specificity for different sugar moieties that are either onto a soluble carbohydrate or onto a carbohydrate moiety that is a part of a glycoprotein or glycolipid (Gemeiner P et al, 2009). Consequently, distinct glycopro files can be generated and compared according to the choice and/or the combination of lectins according to their glycan epitope. A list of lectin that can be used in the methods of the invention includes: GNA (Snowdrop lectin), PNA (Peanut agglutinin), VVL (Hairy vetch lectin), WGA (Wheat Germ agglutinin), SNA (Sambuscus nigra lectin), MAL/MAA (Maackia amurensis leukoagglutinin), MAH (Maackia amurensis hemoagglutinin), LFA (Limax flavus agglutinin) and ConA (Concanavalin A).

The Examples below show that a specific glycan known as sialic acid, alone or in polymerized forms of this glycan known as polysialic acid, as well as any glycan recognized on the basis of the presence of a sialic acid (hereafter collectively defined as "PolySia-related epitopes") can be preferably used in methods for isolating Glyco-MPs populations of interest.

The expression "sialic acid," as used herein, is a generic term for the N- or O- substituted derivatives of neuraminic acid, a nine-carbon monosaccharide (Varki A, 2008). The amino group of neuraminic acid typically bears either an acetyl or a glycolyl group in a sialic acid. It is also the name for the most common member of this group, N-acetylneuraminic acid (Neu5Ac or NANA) and 2-Keto-3- deoxynononic acid (Kdn). Other members of sialic acid include, but are not limited to, N-Acetylglucosamine, N- Acetylgalactosamine (GalNAc), N-Acetylmannosamine (ManNAc) and N-Glycolylneur- aminic acid (Neu5Gc). Sialic acids are found widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. The amino group bears either an acetyl or a glycolyl group. The hydroxyl substituents may vary considerably: acetyl, lactyl, methyl, sulfate and phosphate groups have been found. Hydroxyl substituents present on the sialic acid may be modified by acetylation, methylation, sulfation, and phosphorylation. The predominant sialic acid is N-acetylneuraminic acid (Neu5Ac). Sialic acids impart a negative charge to glycans, because the carboxyl group tends to dissociate a proton at physiological pH.

Glycosylation changes associated with apoptosis, including at the level of sialylation, have been reported in different cell lines from different histological origin (colon, breast, pancreas, and bladder cancer) as these cells are recognized by selected lectins, in presence or in absence of specific enzymes that synthesize or degrade sugar chains (Malagolini N et al, 2009). However, there is no evidence in the literature on any preference or other features of glycosylation that can be used for isolating MPs populations having medical and/or technical relevance.

Moreover, the Examples below show that evidences generated by studying the glycan epitopes present on the surface of cell lines in presence of apoptotic agents cannot be used for interpreting or anticipating the presence of specific glycan epitopes on the surface of Glyco-MPs populations, which rather appear as biological entities providing distinctive (and possibly more relevant) biological information.

Antibodies to variants of PolySia-related epitopes (in terms of chemical derivativization, linkage and/or degree of polymerization) have been generated and compared in terms of binding specificity, epitope presentation, and degree of polymerization (Hayrinen J et al, 1995; Hayrinen J et al , 2002; Sato C et al, 1995; Sato C et al , 2000), establishing the specificity of these antibodies for PolySia related epitopes having defined lengths arid degrees of polymerization ranging from 2 up to 200 sialyl residues. A series of human and rodent proteins have been showed as presenting one or more PolySia-related epitopes and/or degree of polymerization of the PolySia-related epitopes, the main one being PSA-NCAM has been used for identifying, studying the activities, and/or sorting specific cell types such as neuronal cells (Bonfanti L, 2006) and pancreatic beta-cell subpopulations (Banerjee M and Otonkoski T, 2009). Other proteins presenting PolySia-related epitopes have been identified such as betal integrin (Bartik P et al , 2008) and DPP-4 (Cuchacovich M et al. 2001), sodium channels (Zuber C et al , 1992), and CD36 (Yabe U et al, 2003). Moreover, panels of peptides and proteins that are linked with PolySia-related epitopes were identified and quantified in blood using (glyco)proteomic technologies, provding additional candidates of proteins that can be associated to this type of glycosylation (Alley W and Novotny M, 2010; Kurogochi M et al. ,2010).

A list of preferred PolySia-related epitope and related PolySia- binding agents that are known in the literature and then can be used in methods for identifying Glyco-MPs populations is provided in Table 2.

Table 2

The interaction between Glyco-MPs populations and such glycan- binding agents can be studied by means of competing soluble glycans such as those generated using chemical technologies and characterized by mass spectrometry (Galuska S et al , 2007; Patane J et al , 2009). Moreover, technologies for the enrichment of peptides that are linked to glycans can be used for the analysis of the structure and attachment site identification of glycans that are present on the Glyco- MPs populations (Nilsson J et al, 2009). Series of glycan-binding agents may be used for detecting and isolating different types of Glyco-MPs subpopulations in parallel.

However, glycan-binding agents that are compatible with technologies that maintain MPs integrity (such as FACS, immunological assays, or magnetic beads-based cell isolation are preferred. For example, a panel of antibodies and binding proteins that have been characterized in the literature for different PolySia-related epitopes into biological materials can be used for detecting, isolating and comparing the corresponding Glyco-MPs (sub)populations presenting such PolySia-related epitopes.

This analysis can be accompanied also by the detection of known cell type-specific antigens (using antibodies or other molecules that specifically bind such antigens) that can be more or less frequently associated to total MPs populations or Glyco-MPs (sub) populations within biological fluids and/or in cell culture conditions. List of antigens that can be used to further distinguish Glyco-MPs populations according to their cell origin has been published (Burnier L et al, 2009; Orozco A and Lewis D, 2010; van der Heyde H et al, 2011).

The study of Glyco-MPs populations that expose glycan epitopes (such Polysia-related epitopes) can be also performed by using experimental in vitro and in vivo approaches for either inhibiting glycosylation or for integrating unnatural precursors for labelling the glycan epitopes (Bork E et al, 2007). Preferably, the analysis of Glyco-MPs populations comprises detecting alterations in one or more features of sialylation, including the type of linkage, the degree of polymerization, modifications of sialic acids (including sulfation, branching, presence or absence of a bisecting N-acetylglucosamine), and changes in the number of polysialylated proteins, lipids and/or molecule-specific sites on Glyco-MPs. These studies can be performed in parallel with the determination of other features of Glyco-MPs populations such as the presence of cell type-specific antigens or of biological activities that are established in vitro using cell line-based assays (as for procoagulant activity).

The methods of the invention provide Glyco-MPs populations that are separated from the acellular fraction by means of at least one further binding agent recognizing a phospholipid, a protein, a lipid, or a glycan other than the one used for isolating the Glyco-MPs population. This additional binding agent preferably binds a phospholipid which is phosphatidylserine. In particular, said phosphatidylserine- binding agent is Annexin V or Lactadherin (Shi J and Gilbert G, 2003; Logue et al. 2009) or other proteins, peptides, or chemicals (Thapa N et al , 2008; Lemmon M, 2008; Stace C and Ktistakis N et al, 2006).

Phospholipid-binding agents, and in particular phosphatidylserine- binding agents, are preferred, with preference for those not requiring calcium for binding phosphatidylserine and not altering MPs integrity. MPs population have been often isolated and characterized from biological fluids using phosphatidylserine-binding agents that are in a solid or a liquid phase.

Alternatively (or in addition) the methods may involve the use of one or more additional binding agents that bind a protein, lipid, or glycan of the cell surface that is transferred to the surface of Glyco-MPs populations following their release from the cells, thus identifying their origin. These methods can be performed using a binding agent that bind a protein, lipid, or glycan of the cell surface is defined according to a specific cell type, tissue, organ, drug treatment, age, sex, pathology, genotype, phenotype, predisposition, viral infection, and/or clinical status. Additional binding agents to e used according to the invention can be defined as agents that bind a cell type-specific antigen {e.g. , an antibody).

The separation and/or the detection of Glyco-MPs populations with an additional binding agent can be performed prior to, simultaneously, or following the separation step that involve the glycan-binding agent and may be used as well for separating MPs populations. This step may be performed in liquid or solid phase, and in the latter case the solid phase can be in the forms of beads, and in particular magnetic beads, a support that has been already used for immobilizing Annexin V and sorting apoptotic cells from a biological fluid (Said T et al , 2008). These additional binding agents can be used not only as a mean for positively selecting the Glyco-MPs population but as a negative selection tool {e.g., for eliminating specific MPs populations presenting a specific cell surface antigen or cell debris and other undesired entities within the sample of biological fluid). Equally, specific Glyco-MPs subpopulation may be negatively selected in order to define an MPs subpopulation having a potential medical or biological interest For instance, a MPs subpopulation that is deprived of specific glycan epitopes may be enriched in other antigens that may be of interest for defining and validating a biomarker in a population.

A further aspect of the present invention is a Glyco-MPs population that is obtained according to the methods defined above. The Glyco-MPs populations can be provided in a liquid or a solid phase, and in association or not with the binding agent. These novel biological entities can provide novel biomarkers that are associated to Glyco-MPs in general or to specific Glyco-MPs subpopulations of interest that are defined in connection to specific cell types and/or disorders. For instance, such biomarker can allow screening subjects at risk of being affected by a disorder, since it can be identified by using common technologies such as flow cytometry, mass spectrometry, gel electrophoresis, an immunoassay (e.g. , immunoblot, immunoprecipitation, ELISA), nucleic acid amplification, procoagulant activity, and/or electron microscopy on biological fluids or Glyco-MPs populations obtained from such subjects in a singleplex or multiplex formats as summarized in Figures 1 and 5. For instance, the Examples show that a significant fraction of a Annexin V-positive MPs population can be isolated and characterized as a Glyco-MPs population by double staining in flow cytometry.

The Glyco-MPs population provide means for defining novel biomarkers. In particular, the biomarkers can be defined by means of the concentration of Glyco-MPs population only or the concentration of Glyco-MPs and of the concentration of at least another population of MPs that present a protein, lipid, phospholipid, and/or glycan of the cell surface. Such biomarkers can correspond to a peptide, a protein, a phospholipid, a lipid, a nucleic acid, a glycan, or any combinations of such Glyco-MPs components. The biomarker can be specific for a disorder and may be identified by means of one or more technologies such as flow cytometry, mass spectrometry, gel electrophoresis, immunoassay, nucleic acid amplification, or in vitro assays for a biological activity.

The term "biomarker" refers to a molecule, a parameter, a characteristic, or an entity that is objectively measured and evaluated as an indicator of a specific state of an organism, in particular in association to a normal or pathogenic process, or the response to a medical treatment.

In the present case, this factor can be defined by the concentration and/or the components of Glyco-MPs that are isolated from biological fluids of humans or animals (rodents or primates, in particular). Accordingly, the biomarkers can be defined by means of:

a) The concentration of a Glyco-MPs presenting a specific glycan epitope (for example, a PolySia-related epitope);

b) The ratio between the concentration of total MPs population and of a Glyco-MPs population of (a); 6035

c) The ratio between the concentration of a Glyco-MPs population of (a) and a different glyco-MPs population;

d) The concentration of MPs not presenting the specific glycan epitope of (a); and/or

e) The concentration of MPs populations of (a) and/or (d) that present a protein, lipid, phospholipid, and/or glycan other than the specific glycan epitope of (a).

The biomarker can be found associated to the whole Glyco-MPs population and/or to specific Glyco-MPs subpopulations defined by any molecular parameter of interest (for example, the presence of a cell-type specific antigen). The quantitative evaluation of Glyco-MPs in specific volume of a biological fluid can be, or not, associated to a quantitative evaluation of total MPs in such volume. The concentration of Glyco-MPs (sub)populations that present (in particular on the surface) a cell component (e.g. , a protein, a protein variant, a phospholipid, a nucleic acid, a glycan, a glycoconjugate) or any other organic or inorganic elements may be used as biomarker. Such component that is found associated to a Glyco-MPs populations can be used as a biomarker that allows establishing a specific status of the cells originating the Glyco MPs (sub) population and/or the possible interactions of Glyco MPs (sub)populations with the surface of the specific cell types or of a virus, with a drug, an antibody, and any other compound present in the biological fluid.

An association between a biomarker (such as a Glyco-MPs population or a Glyco-MPs component) and a disorder can be established independently from the cause of the disorder but only from its effects and other associated biological evidences.

The Glyco-MPs populations can allow the identification of biomarkers for characterizing the state of a subject (such as normal, affected or at a risk of disorder, responding or not to a therapy) by using samples of one or more biological fluid obtained from such subject. Once that biomarkers are found associated to Glyco-MPs, such biomarkers can be identified in the subjects of interest (e.g., animal models, patients, at risk individuals) for obtaining information of medical interest on a subject, throughout the time (e.g. , before, during, and/or after a medical intervention or treatment) and/or in comparison to reference populations (e.g., control, healthy subjects or subjects affected by a disorder). Such biomarkers may be detectable even without using Glyco-MPs populations but, given the complexity of biological fluids, Glyco-MPs population may provide a more precise and reliable analysis of biomarkers otherwise undetectable. Alternatively, the subtraction of specific Glyco-MPs subpopulation may provide a MPs population that is particularly enriched (or deprived of) specific antigens in defined test conditions, thus representing an alternative mean to identify biomarkers

The present Invention also relates to kits for isolating and/or using Glyco-MPs for medical or veterinary application. The kits for isolating Glyco-MPs populations comprise a glycan-binding agent (for example, specific for a PolySia- related epitope as listed in Table 2) and at least one further binding agent recognizing a phospholipid (for instance, a phosphatidylserine-binding agent) a protein, a lipid, or a glycan other than the one used for isolating the Glyco-MPs population. The binding agents for the desired molecules can be provided in a liquid or a solid phase, with or without means for detecting and comparing effectively the interaction with Glyco- MPs (and consequently for quantifying the Glyco-MPs (sub)population of interest) by using one or more proteomic, immunological, biochemical, chemical, biological or nucleic acid detection method.

The present invention also provides the use of the Glyco-MPs populations, or of a kit as defined above for identifying biomarkers of medical interest in a sample of biological fluid. At this scope, the Glyco-MPs populations of the invention can be isolated, compared, and used according to desired medical application. Examples of the process for analyzing and comparing Glyco-MPs and identifying biomarkers of medical interest are summarized in Figures 1 and 5, but many other possibilities can be envisaged in connection to specific medical goals, features of the biomarker, and/or the type of populations to be evaluated.

In particular, the biological fluids into which Glyco-MPs features are studied, can be obtained from distinct groups of subjects that are appropriately selected (e.g., on the basis of drug treatment, age, sex, pathologies, genotype, phenotype, exposure to risk factors, viral infection, or clinical status) and then compared at the level of Glyco-MPs (sub)populations using biomarkers that can be evaluated by means of one or more proteomic, immunological, biochemical, chemical, biological, or nucleic acid detection method. This comparison may also involve the use of appropriate statistical and/or imaging methods (including MRI, CAT, and ultrasound, immunodiagnostic test, detection of protein levels, or biopsy), should allow confirming the identification of a biomarker associated to Glyco-MPs that can be further used in diagnostic and drug discovery/validation methods for a disorder, as well as of any other disorder that may alter the structure and/or the activity of an organ, a tissue, or a cell type. The present invention also provides methods for diagnosing or monitoring a disorder that comprises the identification of a biomarker that have been characterized using Glyco-MPs. Such medical methods involve the isolation, the characterization, and the comparison of Glyco-MPs populations in a sample of biological fluid. Glyco-MPs quantitative and/or qualitative features might be of considerable value for diagnosing and monitoring of human disorders, as well as for evaluating drug candidates and drug treatments for any disease, and in particular for establishing their effects on biological fluids.

These methods can involve determining Glyco-MPs concentration and/or composition in test and control samples by suing technologies such as flow cytometry, mass spectrometry, gel electrophoresis, an immunoassay (e.g. , immunoblot, immunoprecipitation, ELISA), nucleic acid amplification, procoagulant activity, and/or electron microscopy on biological fluids or Glyco-MPs populations (that is, by applying technologies that allow the identification of biomarkers of interest). These methods may involve the isolation and the comparison of Glyco-MPs populations within selected biological fluids (e.g. , blood, urine) where Glyco-MPs populations can be isolated.

Optionally these methods may involve comparing the concentration and/or composition of total MPs. Still optionally, the methods may involve the detection of the biomarker(s) that can be found associated with Glyco-MPs populations within a tissue. Such tissues can be the ones from which Glyco-MPs populations can be originated (e.g. , obtained from biopsies of the CNS) but can also be any other cell types or biological material of interest for diagnosing or monitoring a disorder.

It will be understood that it is not absolutely essential that an actual control sample be run at the same time that assays are being performed on a test sample. Once "normal" (i. e. control) concentration and/or composition of Glyco-MPs populations (i.e. Glyco-MPs vs plasma MPs population ratios, Glyco-MPs populations presenting or not a specific cell surface antigen) have been established, these levels can provide a basis for comparison without the need to rerun a new control sample with each assay. The comparison between the test and control samples using appropriate statistical methods and criteria should provide a basis for a conclusion on the state of the subject, for instance whether the disorder is progressing or regressing in response to a treatment, if the subject is (or will be) affected by a disorder, or if the subject has been exposed to a drug, to a traumatic insult, or any other event that alters the metabolism within a biological fluid, and in particular the generation of Glyco- MPs populations.

The term "diagnosing" refers to diagnosis, prognosis, monitoring a disorder in a subject individual that either has not previously had the disorder or that has had the disease but who was treated and is believed to be cured. This application of the methods of the invention can be extended to the selection of participants in (pre) clinical trials, and to the identification of patients most likely to respond to a particular treatment.

The term "monitoring" refers to tests performed on patients known to have a disorder for the purpose of measuring its progress or for measuring the response of a patient to a therapeutic or prophylactic treatment.

The term "treatment" refers to therapy, prevention and prophylaxis of a disorder, in particular by the administration of medicine or the performance of medical procedures, for either prophylaxis (prevention) or to cure the infirmity or malady in the instance where the patient is affected.

The quantitative and/or qualitative in vitro/ex vivo analysis of Glyco-MPs populations provide relevant information for evaluating a subject, having a predefined clinical status, disorder predisposition, positive/negative response to a treatment, and/or sensibility to a drug or a pathogen (see Figure 1). In fact, this analysis may lead to a definition of a profile in which different elements characterizing Glyco-MPs populations are used, including concentration (absolute or relative to total MPs or cell type-specific MPs in the preferred biological fluid), presence/absence of one or more antigens, reduced/increased presence of one or more antigens, size, phospholipid composition, intracellular components, and the like. This method of evaluation can be also applied for characterizing a cell population that is maintained in vitro/ex vivo (such as cell lines, primary cells, stem cell, tissue material preparations) whereby the presence of Glyco-MPs population is established in the cell culture supernatant (see Figure 5). In this manner, human Glyco-MPs can be identified and characterized in patients that have been selected by different criteria (for example at risk, suffering, or under treatment for a disorder).

The Examples below demonstrated that, by combining in vitro and in vivo studies of biological fluids, Glyco-MPs populations can be detected, isolated, and characterized for identifying biomarkers of medical interest, in particular by using proteomic and other technologies that are described in the literature for studying specifically membrane proteins within biological fluids (Lai Z et al. , 2009; Cordwell S and Thingholm T, 2010), not only in blood but also in urine (Smalley D et al, 2008a) or cerebrospinal fluid (Hale J et al , 2008; Shi M et al , 2009; Roche S et al , 2008; Zougman A et al, 2008),

All references cited herein are fully incorporated by reference in their entirety. Having now fully described the invention, it will be understood by those of skill in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

EXAMPLES

Example 1; Quantification of Glyco-MPs in the blood of rodent models

Materials & Methods

Preparation of platelet-free plasma from rat blood in the diabetes models

Two different rat models of diabetes were used to determine Annexin V-positive and Glyco-MPs levels in vivo. First, male Zucker diabetic fatty (ZDF) rats (20 weeks old, Charles River, USA) were compared to age-matched Zucker lean controls (ZLC). Second, male Sprague Dawley (SD) rats (8-weeks old, Janvier, France) received for 5 days a daily injection of Streptozotocin (20mg/kg) or saline. At the end of the protocol (for both models), animals were fasted for 6 hours, and then anesthetized using Isoflurane. In these rat models, blood was collected by retro-orbital puncture and transferred into a tube containing 1/10 volume of citrate buffer in order to prepare platelet-rich plasma.

In both cases, within one hour of collection, the blood samples is centrifuged at l,500g for 15 minutes at room temperature. The supernatant is then carefully removed and transferred to a new tube.

Platelet- free plasma is then obtained by centrifugation at 13,000g for 2 minutes at room temperature. Again, the supernatant is carefully transferred into a new tube and snap-frozen using liquid nitrogen. Samples were stored at -80°C until use.

Preparation of platelet-free plasma from mouse blood in the diabetes model

Male Db/db mice (13-weeks old, CERJ, Le Genest Saint Isle, France, 15 animals per group) were compared to their lean counterpart (db lean) in standard chow diet. At the end of the protocol, animals were fasted for 6 hours, and then anesthetized using isoflurane. Blood was collected by retro-orbital puncture and transferred into a tube containing 1/10 volume citrate in order to detect the different MPs populations. Another tube with EDTA was prepared for glucose and insulin analysis. Platelet-free plasma is then obtained and stored as indicated above for samples of rat origin.

Production and purification of recombinant human Annexin V protein

The DNA encoding human Annexin V (Genebank NM_001 154) was used for producing Histidine-tagged, recombinant Annexin V in bacteria (E. Coli strain BL21 star PI OS). The recombinant protein results from the fusion of the DNA sequence coding for a synthetic sequence (MGRSHHHHHHGMASMTGGQQMGRDLYDDDKDRWGSE; SEQ ID NO: 1) that includes an hexahistidine tag (HHHHHH; SEQ ID NO: 2) and the Xpress epitope (DLYDDDK; Invitrogen Life Technologies), in 5' to the DNA encoding human Annexin V (amino acids 1-320). The Histidine-tagged, recombinant Annexin V was purified using an HIS-Trap column (GE Healthcare). Purity was assessed by SDS- PAGE gel and sequence was verified by MALDI-TOF mass spectrometry. His-tagged Annexin V was then labelled with FITC (NHS-Fluoroscein; Thermo-Scientific, Pierce Protein Research Products; Cat. No. 46410), following the manufacturer's protocol.

Quantification of phospholipid-positive, Glvco-MPs, antigen specific populations

MPs populations presenting phosphatidylserine and/or PolySia- related epitopes on their surface were identified in Platelet-free plasma as cell particles having a diameter comprised between 0.5 and Ι μιη and positively stained with labelled Annexin V and/or with indicated lectin or monoclonal antibodies, respectively. In addition, counting beads (Flowcount Fluorospheres, Beckman- Coulter, France; 30μ1 / sample with a 0.5μιη cut-off to obtain reproducible results) were added to each sample in order to express MPs counts as absolute number per μΐ of biological fluid.

The samples of platelet-free plasma (each having a volume of 30μ1) were incubated in the dark for 30 minutes with either His-tagged, FITC-labeled Annexin V (60ng) in incubation buffer (2.5mM CaCl₂, 140mM NaCl, lOmM Hepes pH 7.4) or Annexin V PE-Cy5 that was prepared with the Lightening Link TM PE- Cy5 Tandem Conjugation kit. In the latter case, 60 μg of Annexin V was labeled with Lightening Link Modifying agent with 100 μg of PE-Cy5 3 hours in a dark room.

Distinct Glyco-MPs populations were analysed by flow cytometry using, as glycan-bnding agent, a monoclonal IgM antibody directed to PolySia (final concentration 30 g/ml; AbCys, clone 2-2B; Rougon G et al, 1986) which was then 5

detected using a PE-conjugated secondary antibody (Goat anti-mouse IgM PE, dilution 1/8; Caltag Laboratories) or using a fluorescent lectin from Maackia amurensis (MAA FITC; EY laboratories; Cat. No. F-7801-2). When double staining reactions were performed, the labelling reaction was stopped with a quencher and the preparation was adjusted at 15 μg/ml. 30 μΐ of MPs were mixed with 10 μΐ of Glycan- binding agent at 300 g/ml and Ι ΟμΙ of Annexin V (AnnV PE-Cy5) at 15 μg/ml or PE anti-mouse Ter-1 19 (TER; BD-Pharmingen, Cat. No. 553673) at 20 μg/ml for erythrocyte -derived MPs or PE-Cy7 anti-mouse CD41 (CD41 PC7; eBioscience, Cat. No. 25-041 1-80) at 10 μg/ml for platelet-derived MPs. Samples were then analyzed by flow cytometry.

The quantifications were performed for each sample by FACS (FC500 flow cytometer; Beckman-Coulter, France), using the two MPs-labelling compounds either in parallel on two different aliquots of the same sample, or simultaneously on the same aliquot, then analysing separately the two signals.

Results & Conclusions

The molecular features of potential medical interest that are present in Glyco-MPs populations can be characterized by applying a process in which samples of biological fluid having different origins (e.g. , blood, cerebrospinal fluid, urine; before or after treatment; in control individuals or patients) are isolated and compared for the quantitative and qualitative features using appropriate technologies (Figure 1).

At least initially, the Glyco-MPs populations were obtained and defined by using a process that allow comparing the concentration of glycan-specific and phospholipid-specific MPs populations in different subjects, in particular for assessing the contribution of glycan-specific Glyco-MPs populations to the overall MPs concentration in a sample of biological fluid, as generally defined on the basis of the presence of phosphatidylserine on their surface. This approach was applied for initiating a more general evaluation of quantitative, size, and/or molecular features of Glyco-MPs populations that are obtained using samples of biological fluid isolated from different subjects in conditions that maintain the MPs integrity.

At this scope, a series of experiments were designed to quantify in parallel Annexin V-positive MPs populations and Glyco-MPs populations in blood of both rat and mouse origin by flow cytometry, using fluorescent calibrated beads with a 0.5μπι cut-off to obtain reproducible results (Robert S et al , 2009). Glyco-MPs populations were identified and compared to phosphatidylserine-containing Annexin V-binding MPs populations by means of a commercial anti-PolySia antibody, in order to determine if polysialic acid can be considered as a candidate glycan for isolating and characterizing Glyco-MPs populations.

Blood was collected and samples were processed according to the technical flowchart (Figure 1) for identifying Glyco-MPs within preclinical models. In particular, rat models for diabetes were used, such as ZDF rats and rats injected with a low dose of Streptozotocin for 5 days, in order to induce pancreatic degeneration leading to a decrease in insulin secretion and ultimately to diabetes (Chen D and Wang M, 2005).

Flow cytometry analysis not only confirmed that PolySia-positive Glyco-MPs population can be identified in rat plasma as a substantial fraction (about 60%) of Annexin V-positive MPs population in control samples but also that the levels of both two MPs populations were proportionally increased in both ZDF rats (Figure 2, panels A and B) and Streptozotocin-injected rats (Figure 2, panels C and D), even if the basal concentration of MPs populations in control rats were different in the two models by almost an order of magnitude.

Another rodent model for diabetic dyslipidemia has been used for assessing the potential relevance of Glyco-MPs. The db/db mice are homozygous for a mutation of Leptin receptor that become identifiably as obese around three to four weeks of age. Elevations of plasma insulin begin at 10 to 14 days of age and of blood sugar at four to eight weeks. A number of features are observed in this model, including an uncontrolled rise in blood sugar and severe depletion of the insulin- producing beta-cells of the pancreatic islets, and consequently it is used for preclinical validation of candidate drugs against type 2 diabetes and dyslipidemia (Reifel-Miller A et al, 2005).

In this model, the induction of Annexin V positive MPs reached 1.5- fold in db/db mice (n=13) versus the control group (n=12) including db/lean mice (heretozygous for this specific mutation). This trend is maintained for MPs that are positive for an erythrocyte-specific antigen but not observed in a Glyco-MPs population that is isolated by means of a labeled lectin (MAA). However, if the erythrocyte-specific antigen is detected in the Glyco-MPs subpopulation, this small MPs subpopulations has a 3.6 fold increase in the db/dbmice when compared to controls that is significatively higher than the 2.3-fold induction of MPs subpopulation in dbdb mice that is positive for both Annexin V and the erythrocyte-specific antigen. The platelet fraction of total Annexin V positive population did not detect any change between insulin-resistant and lean animals even if combined with MAA-based detection (Figure 3 A). The development of insulin resistance in db/db mice at 13 weeks in the experiment was confirmed by measuring the glycemia (Figure 3B) and insulinemia (Figure 3C).

Thus, it was surprisingly demonstrated that Glyco-MPs populations, such as positive MPs populations that are positive for PolySia-related epitope, can be isolated and used in parallel with (or in alternative to) known methods, such as those based on phospholipids or cell type-specific antigens, for establishing the qualitative and quantitative features of MPs subpopulations in a biological fluid, for example that is obtained from preclinical models of diseases, such as those for diabetes, endotoxemia, atherosclerosis, cancer, or rheumatoid arthritis.

It can be suggested that, by using binding agents specific for different glycans and/or different biological fluids, different percentages of total MPs populations are identified as Glyco-MPs populations. However, the specific Glyco- MPs populations (such as PolySia-positive Glyco-MPs or Glyco-MPs subpopulations identified according to lectin binding features) are of particular interest. Glyco-MPs subpopulation appear as being generated and metabolized within biological fluids in manners that appears being either very different or very similar to the overall MPs populations (even if such Glyco-MPs populations have almost certainly different cell origins and even if a different cell surface components are known to be modified with glycans) depending on glycan epitope/binding agent (such as PolySia-related epitopes and corresponding binding agents) that is used.

This finding is of major importance since there are no evidence in the literature on if and how the presence of a specific glycans on the surface of MPs allow identifying alternative MPs populations that have relevant features for assessing the overall MPs concentration in a sample of biological fluid, as well as and drug- and/or disease induced changes of potential medical interest, as in the rodent models described in this Example.

Example 2: Quantification of Glyco-MPs in human blood

Materials & Methods

Preparation of platelet-free plasma from human blood in control- case studies

In humans, blood was collected using a classical 19-gauge needle by slow-pull syringe venipuncture and, after discarding the first 2 ml of blood, the subsequent 6 ml of blood transferred into 1/10 volume of 3.2% sodium citrate (Vacutainer Tubes, BD).

In the case-control study for evaluating MPs populations in coronary patients compared to control subjects, two 2 parallel groups (each comprising 6 subjects) were established in open conditions: stable coronary patients (coronarian) versus control subjects.

All participants were Caucasian male aged from 50 to 75 years, in a seemingly good health at the time of selection, and provided written informed consent prior to enrolment. The stable coronary patients were included on the basis of a status defined as "stable coronary disease" that is defined by one or more of the following conditions: stable or unstable angina pectoris with positive ECG stress test or positive myocardial scintigraphy or stenosis of >50% of coronary artery, history of myocardial infarction, history of coronary revascularization, under treatment by Aspirin and/or statin at stable dose for at least 3 months. The control subjects were included as not having a history of coronary disease or family history of coronary disease.

In both cases, within one-two hour of collection, platelet-rich plasma was obtained from the blood samples by centrifugation at l ,500g for 15 minutes at room temperature. The supernatant is then carefully removed and transferred to a new tube. Platelet-free plasma is then obtained by centrifugation at 13,000g for 2 minutes at room temperature. Again, the supernatant is carefully transferred into a new tube and snap-frozen using liquid nitrogen. Samples were stored at -80°C until use.

Quantification of phospholipid-positive MPs and Glyco-MPs populations

Phospoholipid-positive MPs populations presenting phosphatidylserine or Glyco-MPs populations that present a PolySia-related epitope as relevant targets on their surface were identified in Platelet-free plasma as cell particles having a diameter comprised between 0.5 and Ι μηι and positively stained with labelled Annexin V. The samples of platelet- free plasma (each having a volume of 30μ1) were incubated in the dark for 30 minutes with either His-tagged, FITC-labeled Annexin V (60ng) in incubation buffer (2.5mM CaC12, 140mM NaCl, lOmM Hepes pH 7.4), or with a monoclonal IgM antibody directed to a PolySia-related epitope (final concentration 30 μ^ηιΐ; AbCys, clone 2-2B; Rougon G et al , 1986) which was then detected using a PE-conjugated secondary antibody (Goat anti-mouse IgM PE, dilution 1/8; Caltag Laboratories). In addition, counting beads (Flowcount Fluorospheres, Beckman-Coulter, France; 30μ1 / sample with a 0.5μιη cut-off to obtain reproducible results) were added to each sample in order to express MPs counts as absolute number per μΐ of biological fluid. Alternatively, 30 μΐ of Glyco-MPs were labelled with Annexin V PE (Beckman Coulter, 731726) or 5 μΐ and Maackia amurensis lectin (MAA FITC; EY laboratories F-7801-2, CA, USA) 10 μΐ at 300 μ^πιΐ. The analysis was performed for each sample by FACS (FC500 flow cytometer; Beckman-Coulter, France), using the two MPs-labelling compounds in parallel, or simultaneously on the same aliquot, then analysing separately the two signals.

Results & Conclusions

A confirmation to the observations that were made in rodent models was sought in samples of human origin, wherein the Glyco-MPs populations were determined by flow cytometry, in absolute terms or combined with Annexin V binding data.

In a first study, PolySia Glyco-MPs populations were identified and compared to phosphatidylserine-containing, Annexin V-binding MPs populations by flow cytometry using the plasma of six individuals. As in the animal model, the levels of these two MPs populations showed an extremely tight correlation (with about 60% of Annexin V-positive MPs that are PolySia-positive Glyco-MPs) in all the samples, even if the basal level of MPs populations were different (Figure 4, Panel A).

In a second study, a group of human subjects were selected according to their clinical status associated to Coronary Artery Disease. This disorder is the main death cause in the industrialized world and is mainly due to atherosclerosis, which is a chronic inflammatory disease, initiated and propagated by continuous damage to the vascular endothelium, referred to as endothelial dysfunction. Apoptosis is a process of cell death that has been shown to occur during the progression of human atherosclerotic plaque. The consequences of apoptosis are deleterious because it enhances plaque thrombogenicity after plaque rupture and leads to acute ischemic events and infarction. Such mechanism is associated with a systemic pro-inflammatory state, with changes in multiple cytokines and shedding of MPs population in blood circulation that are originated from diverse apoptotic or activated cell types. The levels of circulating MP (sub)populations are positively correlated with the development of number of chronic inflammatory diseases including cardiovascular diseases (Mayr M et al, 2008; Boulanger C et al. 2006; Shantsila E, 2009).

In a case-control study that was performed in human subjects, the case group defined as stable coronary patients showed a decrease in the concentration of Annexin V-positive MPs population. However, the Glyco-MPs subpopulation that was selected by using both a lectin (MAA) and Annexin V was actually is induced by 3.2 fold in the case group, wherein this specific Glyco-MPs population that represented less than 2% of Annexin V-positive MPs population in the control group, represented almost 10% (Figure 4, Panel B). 6035

Thus, rodent and (more importantly) human plasma contains detectable amounts of Glyco-MPs populations that can be isolated and characterized in conditions that maintain MPs integrity and that can provide a different understanding of MPs relevance in patho-physiological mechanisms and/or in the definition of biomarkers. Thus, Glyco-MPs subpopulations can be compared and used (as such or as proxy) for establishing novel biomarkers within biological fluids that reflecting a patho-physiological state of a subject and therefore could be used for disease prediction, diagnosis, prognosis and follow-up, with a strong focus on both chronic conditions (including inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, diabetes, cancer, and in general metabolic and cardiovascular diseases) and acute conditions (including transplantation, stroke, sepsis, traumatic injuries).

Example 3: Quantification and characctrization of Glyco-MPs subpopulations that are generated in cell culture conditions.

Materials & Methods

Cell culture protocols

The murine pancreatic cell line Min6 (Miyazaki et αί, 1990) was cultured in DMEM medium (4.5g/L Glucose, Gibco) supplemented with 15% fetal bovine serum (AbCys), 1 % penicilin/streptomycin (VWR, val de Fontenay, France), 1% sodium pyruvate (Gibco), and 3.75 μ^Ι of beta-mercaptoethanol. Cells were cultured at 37°C in 5% C02 atmosphere and seeded either at 35,000 cells/cm2 (for stimulation experiments) or at 5 ^χ 106 into 150 cm2 culture flask (BD Bioscience; for FACS experiments). When cells reached confluence, they were rinsed twice and cultured with serum-free and glucose-free medium. For proteomic experiments, the cells were then treated for 24 hours with palmitate at two concentrations (1.0 mM or 1.5 mM; Sigma, France) or vehicle only (BSA 30% alone) in the same medium.

Human Umbilical Vascular Endothelial Cell (HUVEC) cells (CC- 2517; Cambrex, Walkersville, USA) were cultured in EBM®, Endothelial Basal Medium (EBM media; Cambrex, CC-3121 ; Walkersville, USA) and EBM plus SingleQuots of growth supplements (EGM BulletKit, Cambrex CC-3124; Walkersville, USA). Cells were seeded at 2 500 to 5 000 cells / cm² into 150 cm² culture flask (BD Bioscience; Le Pont-De-Claix, France) and maintained at 37 °C in 5% C02 atmosphere. Cells were used for experiments when they reached 80% confluence. HUVEC were then treated for 24 hours at 37°C without (control condition) or with tumour necrosis factor-alpha (10 μg/ml; R&D System) as apoptotic agent in the culture medium. The human neuroblastoma cell line SH-SY5Y (CRL-2266; ATCC, VA, USA) were plated into 150 cm² culture flask (Falcon Becton Dickinson, Heidelberg, Germany) at 60 000 cells/ cm2 and incubated in MEM culture medium (Gibco), supplemented with Ham's F12 glutamax, 1% penicillin and streptomycin, 0.5% non essential amino acids, 0,5% sodium pyruvate and 10% fetal bovine serum and maintained at 37°C in 5% C02 atmosphere. After having reached 80% confluence, cells were differentiated by culturing them with differentiation culture medium (MEM supplemented medium containing 5 μΜ retinoic acid and 2% fetal bovine serum) for 6 days and then, after washing the cells, with differentiation culture medium supplemented with 10 μg/ml of Brain-Derived Neurotrophic Factor for additional 5 days. Differentiated SH-SY5Y cells were then cultured for 24 hours at 37°C with MEM supplemented medium and DMSO 0.1% (control condition) or MEM supplemented medium with DMSO 0.1% (control condition) and 6-OHDA (defs 100 μΜ) or MEM supplemented medium with DMSO 0.1% and staurosporine (0.5 μg/ml; R&D System) as apoptotic agent.

The immortalized mouse microglial cell line BV-2 (Blasi E et al , 1990) were cultured in RPMI 1640 (Gibco) containing 1.1% GlutaMax, 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were seeded at 2500 to 5000 cell/cm² into 150 cm² culture flask (BD Bioscience) and maintained at 37°C in 5% C02 atmosphere. Cells were washed twice with warm RPMI and then cultured for 24 hours at 37°C in the same medium supplemented with 0.1% DMSO (control condition) or with 0.1% DMSO and Lipopolysaccharides (100 ng/rriL) as apoptotic agent.

The human colon carcinoma cell line HT29 (Cat. No. HTB-38; ATCC, USA) were cultured in McCOY's 5A medium 10% fetal calf serum (FCS), 1 % penicillin/streptomycin (PS) and 1 % glutamine (Invitrogen/Gibco, Cergy Pontoise, France). When cells reached 80% of confluency, they were rinsed twice and cultured for 24 hours at 37°C with 2% serum replacement supplement (SR-3; Sigma, MO, USA) instead of FCS, and without (control condition) or with staurosporine (0.1 μΜ) as apoptotic agent.

The human hepatocellular carcinoma cell line HepG2 (Cat. No. HB- 8065; ATCC, USA) were cultures in DMEM medium 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PS) and 1% glutamine (Invitrogen/Gibco, Cergy Pontoise, France). When cells reached 80% of confluence, they were rinsed twice and cultured for 24 hours at 37°C with 2% serum replacement supplement (SR-3; Sigma, MO, USA) instead of FCS, and without (control condition) or with camptothecin (10 μΜ) as apoptotic agent.

Analysis of cell and MPs populations by flow cytometry

At the end of treatment, cells and MPs populations were analyzed separately.

In the case of cells, after removing the cell culture supernatant, the cells were trypsined and counted. 500 000 cells were analyzed by flow cytometry using Annexin V (FITC or PE) and the glycan-binding agents: Maackia amurensis (MAA FITC; EY laboratories, CA, USA), 2-2B antibody, or KM93 (anti-sialyl Lewis X, 10μg/ml; VWR International, Fontenay sous Bois, France). Ι ΟΟμΙ of cells and 10 μΐ of each glycan-binding agent at 50μg/ml were mixed, spined 5 minutes at 450g to remove the excess of glycan-binding agent and analyzed by flow cytometry. When needed, the following secondary antibodies were used, goat anti-IgG-PE (Abeam; Cat. No. ab97070), goat anti-mouse IgM-PE (Invitrogen; Cat. No. M31504), rat anti- mouse IgG2A-FITC (BD Pharmingen; Cat. No. 553390).

In the case of MPs populations, cell culture superaatants were collected and spun down for 15 minutes at l ,500g at room temperature. Total MPs were then isolated by ultracentrifugation at 30,000g for 45 minutes at room temperature. Supernatants were discarded and pellets were re-suspended in PBS buffer (final volume of 400 microliter per condition). Samples were stored at -80°C until use. Distinct MPs populations were analysed by flow cytometry using Annexin V PE (Beckman Coulter, 731726) and the following glycan-binding agent: fluorescent lectins from Maackia amurensis (MAA FITC; EY laboratories, CA, USA; 20 μΐ of MPs were mixed with 10 μΐ of lectin at 50 μg/ml), 2-2B antibody (IgM)-PE (MACs, 130-093-274), or KM93 (anti-sialyl Lewis X, Ι ΟμΙ at K^g ml; VWR International, Fontenay sous Bois, France).

Results & Conclusions

The composition of biological fluids depends not only from the activity of cells circulating within or surrounding the biological fluid (e.g., endothelial cells or platelets, leukocytes, in the case of blood) but also from the drainage of the interstitial liquid of the organs and tissues. Thus, biological fluids (and consequently MPs populations within them) may be therefore considered as a reporter of the pathophysiological status of large variety of cell types for which specific biomarkers can be identified into MPs populations, or more specifically Glyco-MPs populations that are associated to specific cell types, biological activities, exposure to chemicals, and/or diseases. The molecular features that allow differentiating cell type- and/or disease-specific Glyco-MPs (sub)populations of potential major interest can be initially defined by means of specific primary cells or cell lines that are related to the disease which and that can be used to generated Glyco-MPs within cell culture, in more tightly controlled conditions than in physiological biological fluids (Figure 5). However, on the basis of the findings of Example 1 , the presence and metabolism of cell type-specific, PolySia-positive Glyco-MPs populations can be also evaluated to understand if and how its ratio with Annexin V-positive MPs populations changes between cell types, and thus if different cell type contributes in different manner to the PolySia-positive Glyco-MPs populations.

A preliminary study was performed in a murine insulinoma cell line (Min6) that was stimulated with increasing concentrations of palmitate, a compound that induces stress and apoptosis in pancreatic beta-cells (Jeffrey et al, 2008). At the end of the treatment, the cell culture medium was collected and spun down and MPs populations were then quantified by flow cytometry. Min6 cells appeared to generate significant levels of both Annexin V-positive and Glyco-MPs populations in serum-free culture medium but the basal level of MPs populations was further increased b the exposure to palmitate in a significant and dose-dependent increase for both MPs populations with a similar dynamics. However, if Glyco-MPs populations that are characterized as presenting a PolySia-related epitope defined by a monoclonal antibody increases in parallel with the Annexin V-positive MPs populations in the different conditions, its percentage stays around 40%, and not 60% as determined in a cell-type independent manner within human and rat plasma (Figure 6 A and 6B).

The release of Annexin V-positive MPs populations that are also positive for different PolySia-related epitopes was then tested in other cell lines that shed low levels of MPs in normal culture condition and in response to different apoptotic agents. This comparison is intended to verify if these cell lines release distinct Glyco-MPs populations at different rates.

In a first set of experiments, the percentage of Annexin-V positive MPs population that is also either 2-2B antibody- or MAA-positive was calculated, showing these glycan-binding agents allow identifying Glyco-MPs populations that are cell- and/or apoptotic agent-specific (Figure 7, Panel A). Depending on the chosen combinations, the Glyco-MPs subpopulations correspond to almost to the totality of Annexin V-positive MPs (as in the case of SH-SY5Y cells that are exposed to 6- OHDA with 2-2B antibody as glycan-binding agent), are different depending on the selected apoptotic agent (as in the case of SH-SY5Y cells), or present major differences between glycan-binding agents that recognized distinct PolySia-related antigens (as in the case of HT29 and HepG2 cells).

The comparisons of glycan epitopes between control and apoptosis- induced Glyco-MPs subpopulations were performed by flow cytometry but also extended to the original cell lines with the major differences between glycan-binding agents, including those previously used and a monoclonal antibody recognizing another variant of PolySia-related epitopes (KM93, see Table 2; Figure 7, Panels B and C). In fact, the apoptotic agent does not change in a significant manner neither the number of cells that present the specific PoySia-related epitopes on their surface nor the percentage of M93 -positive, Annexin V-positive MPs subpopulation, since this Glyco-MPs subpopulation is increased in a manner similar to the Annexin V-positive MPs population, suggesting that KM93 may almost represent an alternative to Annexin V for detecting MPs populations from specific cell types such as HT29 and HepG2 cells. The situation that is observed using the other agents is different, since there is an overall decrease in the percentage of these Glyco-MPs subpopulations. Actually, they grow much less than Annexin V-positive MPs population (as shown by measured fold induction), suggesting that, at least in specific situations, a Glyco-MPs population may be subtracted from the total MPs population for assessing specific biomarkers in the resulting, more informative MPs subpopulation.

These data confirm that specific Glyco-MPs subpopulations can provide substantially different information on how different apoptotic and other pathophysiological events affect MPs shedding and/or metabolism, and on how biomarkers can be found characterized as being associated to MPs subpopulations (being Glyco- MPs subpopulations or MPs subpopulations not presenting specific glycan epitopes). Moreover, the information on such apoptotic and other patho-physiological events that have been characterized by studying cell surface and composition cannot be equally applied to MPs surface and composition since shedding, metabolic and other biological mechanisms can clearly distinguish them from the originating cells.

It can be concluded that a variety of cell types release Annexin V- positive and MPs populations having specific post-translational profiles (e.g. , glycosylation profiles that characterize Glyco-MPs) but in very variable absolute and relative amounts. When these two MPs populations are compared in biological fluids such as plasma or cerebrospinal fluid, Glyco-MPs populations should provide a representation of MPs-producing cell types different from those of phoshatidylserine- exposing MPs population that are normally selected from biological fluids using Annexin V binding properties. Thus, Glyco-MPs populations (and in particular Glyco-MPs populations that are positive for PolySia-related epitopes) are biological products that are different from those used so far for studying MPs and that provide novel opportunities to identify MPs-associated biomarkers, in particular using proteomic or immunological technologies. Glyco-MPs populations can be used for studying the presence of cell-type specific antigens that are specifically associated to this MPs population, and consequently Glyco-MPs populations can be of even greater medical interest for evaluating the clinical status of a patient, the progression of a disease or the therapeutic efficacy of a drug treatment.

Example 4: MPs glycoproteome and Glyco-MPs

(glyco)proteome

Materials & Methods

Two-dimensional gel electrophoresis

MPs population were obtained from palmitate-induced Min6 cells as indicated in Example 2. Following the step of ultracentrifugation, total MPs populations were used for preparing an extract containing approx. 450 micrograms of protein into 0.5 ml of 2D lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 20 mM spermine base, 0.8% DTT, 0.2% pH 3-10 BioLytes, phosphatase inhibitor cocktails 1 & 2, and EDTA-free protease inhibitor cocktail tablets). The proteins were fully extracted on ice by sonication with 20 second pulses at 20-25%) of maximum amplitude level. This step was repeated 5 times with a minimum of 1 minute pauses. Extracts were centrifuged at 16, lOOg for 10 minutes at 10°C to remove insoluble particles. Total protein content was determined using the 2D-Quant Kit (GE Healthcare). Non-protein impurities were removed using the ReadyPrep 2D Cleanup kit prior to isoelectrofocalisation (IEF) steps. Protein pellets were solubilized by incubating them with 450μ1 of rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 0.4%) DTT and 0.2%> pH 3-10 BioLytes) at room temperature for 5 minutes.

After a further centrifugation at 16, 1 OOg for 5 minutes at 22°C to remove insoluble fragments, samples were subjected to IEF using premade 24 cm IPG strips, nonlinear pH 3-10, on an IPGphor instrument (Bio-Rad Laboratories, Hercules, CA, USA) in electrophoresis buffer (7M urea, 2M thiourea, 4% CHAPS, 0.4% DTT and 0.2% pH 3-10 BioLytes) at 20°C for a total of 140 kVh achieved with a maximum tension of 8000V. After a low current 500V final step, strips were refocused at 8000V for 30 minutes.

Prior to the second dimension, proteins in strips were reduced and alkylated by using 10 mL of equilibration buffer (6M urea, 2% SDS, 50 mM Tris-HCl H 8.8, 20% glycerol, 2% DTT for 15 min and then for 15 minutes with the same buffer without DTT and containing 2.5% iodoacetamide). Equilibrated IPG strips were shortly soaked in 0.22 μm-flltered electrophoresis buffer and immediately transferred onto 20.2x25.5 cm in size and 1 mm thick gradient 8-16% polyacrylamide gradient gels (38: 1 acrylamide:bis ratio) pre-casted into low-fluorescence glass plates treated with bind and repel silane and including 2 fluorescent markers (Jule Inc, CT, USA). Strips were immobilized by embedding them into a 0.5% agarose solution in electrophoresis buffer that was labelled with trace amounts of bromophenol blue. The second dimension separation was performed overnight at 30°C using an Ettan Dalt six device with an updated upper buffer chamber (GE Healthcare) according to the manufacturer's instructions.

After electrophoresis, gels were fixed and then stained for glycoproteins using the ProQ Emerald 488 staining reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Images were captured using a Typhoon 9400 laser scanner (Molecular Dynamics, Sunnyvale, CA, USA) using the 488 nm excitation wavelength with a 100 μιη resolution. Upon data acquisition, gels were briefly rinsed using milliQ water and were then stained for total proteins by an overnight incubation in SYPRO Ruby solution (Invitrogen, Carlsbad, CA, USA). After extensive washings, image acquisition was performed at a 100 μηι resolution using the 457 nm excitation wavelength. DeCyder 6.5 software (GE Healthcare).

Results & Conclusions

A preliminary characterization of the MPs-associated proteome was performed in Min6 cells that, as shown in Example 3, produce substantial amounts of both Annexin V-positive and Glyco-MPs populations that are positive for a PolySia- related epitope. Protein extracts were generated by using total MPs populations and subjected to two-dimensional gel electrophoresis.

Total proteins and glycoproteins were visualized by staining the gel using SYPRO Ruby and ProQ Emerald 488 staining reagents, respectively. This approach allows the fluorescence-based detection of low nanogram amounts of glycoproteins and proteins, thus providing a comparative analysis of the (glyco)proteomes of cell lines and their changes following, for example, the exposure to a growth factor (Hill J et al. , 2009).

As expected, total protein staining revealed the presence of a large number of proteins, many of them glycosylated as detected by ProQ Emerald 488 staining (Figure 7). This proteomic approach can be applied to compare different MPs populations of interest such as Annexin V-positive and Glyco-MPs populations isolated from the same biological fluid, or Glyco-MPs populations that are positive for PolySIa-related epitope and that are originated from different biological fluids (or from the biological fluid of subject treated or not with a drug). The resulting proteomic maps can be then analyzed to identify candidate biomarkers (presenting or not a glycan) using different technologies that are specific for cell surface glycans, membrane microdomains, and/or membrane proteins (Lai Z et al, 2009; Cordwell S and Thingholm TE, 2009; Apweiler R et al , 2009a; Apweiler R et al , 2009b; Zheng Y and Foster L, 2009) in order to define the surfaceome of Glyco-MPs populations as it has been proposed for human cells (da Cunha J et al. ,2009). Moreover this approach can be used to establish if and how Glyco-MPs and total MPs populations are differentially metabolized within the biological fluid, expose cell surface components in specific manners, and/or interact differentially with cells and other components of the biological fluid.This analysis can be performed also by using the extensive lists of glycans and of the corresponding glycan-binding agents (such as such as monoclonal antibodies, plant lectins, and chemicals) that have been published (see Table 1 and Table 2), indicating the specificity of the interaction, the compatibility with the integrity and viability of cells, and their use for identifying specific cell types and/or antigens of medical interest.

Such glycan-binding agents may be used for detecting and isolating different types of Glyco-MPs subpopulations in parallel. However, glycan-binding agents that are compatible with technologies that maintain MPs integrity (such as FACS, immunological assays, or magnetic beads-based cell isolation) should be preferred. For example, a panel of antibodies and binding proteins have been characterized in the literature for different PolySia-containing biological materials and can be used for detecting, isolating and comparing different PolySia-positive Glyco- MPs (sub)populations presenting higher or lower specificity for (un)modified PolySia groups, molecules containing PolySia groups with different degrees of polymerization and/or linkage. This analysis can be accompanied also by the detection of known cell type-specific antigens (using antibodies or other molecules that specifically bind such antigens) that can be more or less frequently associated to total MPs populations or Glyco-MPs (sub) populations within biological fluids and/or in cell culture conditions (Burnier L et al , 2009; Orozco A and Lewis D, 2010; van der Heyde H et al, 201 1). REFERENCES

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Claims

1. A method for identifying Glycan-containing Microparticles (Glyco-MPs) populations comprising the following steps:

a) Obtaining a sample of a biological fluid sample from a subject; b) Isolating the acellular fraction of said biological fluid;

c) Separating the Glyco-MPs populations from the said acellular fraction by means of an agent binding a PolySia-related epitope and at least one further binding agent recognizing a phospholipid, a protein, a lipid, or a glycan other than said PolySia-related epitope.

2. The method of claim 1 wherein the Glyco-MPs populations having a size comprised between 100 and 1000 nanometrs are separated from said acellular fraction.

3. The method of claim 1 or 2 wherein the PolySia-related epitope is polysialic acid.

4. The method of claims 1 to 3 wherein the biological fluid is any of the following: plasma, blood, cerebrospinal fluid, urine, synovial fluid, bronchoalveolar lavage fluid, seminal fluid, saliva, pulmonary edema fluid, and cell culture supernatants.

5. The method of claims 1 to 4 wherein the Glyco-MPs populations are separated from said acellular fraction by means of the further binding agent that recognizes a phospholipid.

6. The method of claim 5 wherein the phospholipid is phosphatidylserine.

7. The method of claim 1 to 4 wherein said protein, lipid, or glycan is a protein, lipid, or glycan of the cell surface.

8. The method of claim 7 wherein the protein, lipid, or glycan of the cell surface is defined according to a specific cell type, tissue, organ, drug treatment, age, sex, pathology, genotype, phenotype, predisposition, viral infection, and/or clinical status.

9. The method of any of the preceding claims wherein the binding agent is an antibody, a protein, a peptide, a lectin, a glycan, a nucleic acid, a lipid, a phospholipid, or an inorganic compound.

10. The method of any of the preceding claims wherein the binding agent is immobilized on a solid phase and/or is labelled.

1 1. A Glyco-MPs population obtained according to the methods of claims 1 to 10.

12. The Glyco-MPs populations of claim 1 1 , wherein said Glyco- MPs are provided in a liquid phase.

13. The Glyco-MPs populations of claim 11, wherein said Glyco-

MPs are immobilized on solid phase.

14. Biomarker that is defined by means of Glyco-MPs populations of claim 1 1 to 13.

15. The biomarker of claim 14, wherein said biomarkers is defined by means of:

a) The concentration of Glyco-MPs presenting a PolySia-related epitope;

b) The ratio between the concentration of total MPs population and of a Glyco-MPs population;

c) The ratio between the concentration of a Glyco-MPs population of (a) and a different glyco-MPs population;

d) The concentration of MPs not presenting a specific glycan epitope of (a); and/or

e) The concentration of MPs populations of (a) and/or (d) that present a protein, lipid, phospholipid, and/or a glycan other than the specific glycan epitope of (a).

16. The biomarker of claim 14, wherein said biomarker corresponds to a peptide, a protein, a phospholipid, lipid, a nucleic acid, a glycan, or combinations of said molecules that are present on the surface of Glyco-MPs populations.

17. The biomarker of claims 14 to 16, wherein said biomarker is specific for a disorder.

18. The biomarker of claimsl4 to 17, wherein said biomarker is identified by means of flow cytometry, mass spectrometry, gel electrophoresis, immunoassay, nucleic acid amplification, or in vitro assays for a biological activity.

19. Kit for isolating the Glyco-MPs of claims 1 1 to 13 comprising an agent binding a PolySia-related epitope and at least one further binding agent recognizing a phospholipid, a protein, a lipid, or a glycan other than a PolySia-related epitope.

20. Use of the Glyco-MPs of claims 1 1 to 13 or of a kit of claim 19 for identifying biomarkers of medical interest in a sample of biological fluid.

21. A method for diagnosing or monitoring a disorder that comprises the identification of a biomarker of claims 14 to 18 in a sample of biological fluid.