Biomarkers for the assessment of post-hemorrhagic stroke

Abstract

Methods of measuring the effects of an intracerebral hemorrhage are disclosed herein and include: acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage, utilizing the at least one blood, serum, or plasma sample to isolate at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least one surface associated biomarker, wherein the at least one surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof, and quantifying the at least one surface biomarker to monitor post-hemorrhage cellular and molecular cascades in the patient.

Description

[0001] Finlayson Docket No.: 2200-1405

[0002] BIOMARKERS FOR THE ASSESSMENT OF POST-HEMORRHAGIC STROKE

[0003] This United States Utility Application claims priority to United States Provisional Patent Application Serial No.: 63 / 703859 filed on October 4, 2024 and entitled “Blood Biomarkers for the Assessment of Post-Hemorrhagic Stroke”, which is commonly owned and incorporated in its entirety by reference.

[0004] FIELD OF THE SUBJECT MATTER

[0005] The field of the subject matter is biomarkers for the assessment of post-hemorrhagic stroke and their uses in various applications.

[0006] BACKGROUND

[0007] Intracerebral hemorrhage (ICH) is a form of stroke, accounting for ~15-20% of all strokes but with mortality rates approaching 50%. The acute rupture of cerebral vessels triggers primary brain injury from the hematoma and mass effect, followed by a cascade of secondary injury processes including excitotoxicity, edema, oxidative stress, and robust neuroinflammation. In particular, ICH elicits an intense inflammatory response around the hematoma, characterized by activation of resident microglia and influx of peripheral immune cells (neutrophils, monocyte-derived macrophages, lymphocytes) into the brain. Among the early inflammatory mediators, interleukin-1 [3 (IL-1 (3) plays a central role. IL-1 p is rapidly upregulated within hours after ICH and is predominantly produced by activated microglia / macrophages, where it exerts potent neurotoxic effects. In experimental models, blocking IL-1 signaling (e.g. via IL-1 receptor antagonist) attenuates edema, blood-brain barrier disruption, and neuronal injury, underscoring IL-i p’s contribution to ICH-induced damage. In clinical studies, elevated levels of inflammatory cytokines in the bloodstream have been associated with more severe hemorrhages and worse outcomes - for example, higher IL-6 concentrations correlate with larger hematoma volumes and greater perihematomal edema. Perihematomal edema Finlayson Docket No.: 2200-1405 regulation also requires water balance by astrocyte water channels, aquaporins, among which aquaporin 4 (AQP4) is the chief modulator of both cytotoxic and vasogenic edema. Animal models demonstrate the role of AQP4 in increasing water permeability to promote astrocyte swelling in cytotoxic edema, with reductions in cytotoxic edema and improvement in neurological outcomes in mice after AQP4 knockout. In vasogenic edema, while blood brain barrier (BBB) breakdown is the primary mechanism, delayed AQP4 upregulation in the edema resolution phase suggests a role in clearance of accumulated interstitial water.

[0008] Blood-based biomarkers lack specificity for central nervous system (CNS) injury, since peripheral inflammation or systemic stress can also elevate these cytokines. This limitation has motivated the search for more brain-specific biomarkers that reflect the cellular events unfolding after ICH. One emerging strategy to achieve CNS-specific biomarker detection is the analysis of neuron-derived and astrocyte-derived exosomes - small extracellular vesicles (EVs -30-150 nm) originating from brain cells that can be isolated from the peripheral blood. EVs are released by virtually all cell types and carry molecular cargo (proteins, lipids, RNAs) characteristic of their cell of origin. Notably, EVs released in the brain can traverse the blood-brain barrier and remain detectable in circulation. This unique feature enables exosome-derived markers to serve as a “liquid biopsy” of ongoing CNS processes. Moreover, the lipid bilayer membrane of exosomes protects their cargo from enzymatic degradation, conferring high stability to biomarkers enclosed within them.

[0009] By immunocapturing EVs that bear both a pan-EV marker (such as CD9) and cellspecific surface markers, one can enrich for brain-cell-specific exosome populations. For instance, exosomes co-expressing CD9 and the neuronal adhesion molecule L1 CAM are regarded as neuron-derived exosomes (NDEs), while those positive for CD9 and the astrocyte marker EAAT1 (GLAST) are classified as astrocyte-derived exosomes (ADEs). EVs expressing CD9 and the oligodendrocyte and co-expressing MOG are regarded as oligodendrocyte-derived exosomes (ODEs). Finlayson Docket No.: 2200-1405

[0010] Measuring pathophysiologically relevant proteins within or on these subsets offers a window into neuron- or astrocyte-specific or oligodendrocyte-specific injury responses. In principle, each biomarker encapsulated in such exosomes may reflect a distinct cellular event in ICH. For example, IL-1 [3 associated with brain-derived exosomes might indicate local neuroinflammatory activation of glia, whereas other local brain proteins could signify neuron injury, astrocyte stress, or oligodendrocyte injury or repair mechanisms.

[0011] Recent studies support the utility of this exosome-based approach in stroke and hemorrhagic injury. Previous work has demonstrated that plasma astrocyte-derived EV levels rise significantly in patients during the first month after acute ischemic stroke, whereas neuron- and oligodendrocyte-derived EVs do not show such increases. Notably, astrocytic exosome levels were especially elevated in stroke patients who developed hemorrhagic transformation of their infarct, suggesting that glia-derived exosomes can report ongoing blood-brain barrier breakdown and hemorrhagic injury in the CNS. In the context of primary ICH, emerging evidence indicates that circulating EV signatures correlate with disease severity and outcomes. For example, one study found that the concentrations of circulating EVs (both large and small) were predictive of longer hospital / ICU stays and worse 90-day functional status (higher modified Rankin Scale scores) in ICH patients. This highlights the promise of exosomal markers in prognostication.

[0012] Building on this paradigm, we hypothesize that measuring IL-1 [3 associated with neuron-derived and astrocyte-derived exosomes will provide a sensitive and specific indicator of neuroinflammation in the brain after ICH. Therefore, by capturing CNS- derived exosomes that have associated IL-1 (3, we may hereby ty locally generated cytokine to brain derived exosomes at the site of inflammation. With this approach we are able to detect relevant biomarkers and molecular signals occurring at the site the post-hemorrhagic stroke and the inflammatory cascade in the brain. Assessment of such exosomal biomarkers associated with brain derived exosomes will yield insight into cellular-level injury processes and potentially improve the specificity and stability of ICH biomarker monitoring. Ultimately, this exosome-based biomarker approach may enhance our ability to track ongoing brain injury non-invasively and to relate molecular changes to Finlayson Docket No.: 2200-1405 clinical endpoints in ICH patients, paving the way for more tailored prognostic and therapeutic strategies.

[0013] Finlayson Docket No.: 2200-1405

[0014] SUMMARY OF THE SUBJECT MATTER

[0015] Methods of measuring the effects of an intracerebral hemorrhage are disclosed herein and include: acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage, utilizing the at least one blood, serum, or plasma sample to isolate at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least one surface associated biomarker, wherein the at least one surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof, and quantifying the at least one surface biomarker to monitor posthemorrhage cellular and molecular cascades in the patient.

[0016] Methods of measuring the effects of brain trauma and degree of recovery are also disclosed herein and include: acquiring at least one blood, serum, or plasma sample from a patient suffering from a brain trauma, neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least one surface associated biomarker, wherein the at least one surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof, and quantifying the at least one surface biomarker to monitor a cellular cascade and a molecular cascade in the patient.

[0017] Additional contemplated methods of measuring the effects of an intracerebral hemorrhage also include: acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage, utilizing the at least one blood, serum, or plasma sample to isolate at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular Finlayson Docket No.: 2200-1405 vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least a first surface biomarker and at least a second biomarker, wherein the first surface associated biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof and wherein the second surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof and is also a coagulation biomarker, a fibrinolysis biomarker, an inflammation biomarker, or a combination thereof, and quantifying the first surface biomarker and the second biomarker to monitor post-hemorrhage cellular and molecular cascades in the patient.

[0018] Finlayson Docket No.: 2200-1405

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Contemplated target biomarkers are found in Figure 1 and disclosed herein. Contemplated methods of monitoring hemorrhage are disclosed herein that include: a) providing a biological sample; and b) detecting double positive signals of both exosome or extracellular vesicle markers and coagulation or fibrinolysis markers.

[0021] Figure 2 shows plasma samples that were applied to anti-CD171 -immobilized ELISA wells to capture NDE. After removing unbound materials from ELISA wells, wells were reacted with biotinylated antibodies against CD9 (Fig. 2 left) for the quantification of captured NDE, ILIbeta (Fig. 2, 2ndfrom left) for the analysis of neuroinflammation, BDNF (Fig. 2, 2ndfrom right) for the analysis of brain’s recovery function, and UCHL-1 (Fig. 2, right) for the analysis of neuronal cell death, respectively. Biotinylated antibodies were further reacted with streptavidin-horseradish peroxidase, followed by chemiluminescent substrate, SuperSignal, and relative light units (RLU) were determined by a luminometer. Y-axis: % from the Day 1 values (200% means double from the Day 1 ). X-axis: Days after stroke.

[0022] Figure 3 shows plasma samples that were applied to anti-EAAT1 -immobilized ELISA wells to capture ADE. After removing unbound materials from ELISA wells, wells were reacted with biotinylated antibodies against CD9 (Fig. 3 left) for the quantification of captured ADE, GFAP (Fig. 3, middle) for the analysis of astrocyte conditions, and AQP4 (Fig. 3, right) for the analysis of brain edema, respectively. Biotinylated antibodies were further reacted with streptavidin-horseradish peroxidase, followed by chemiluminescent substrate, SuperSignal, and relative light units (RLU) were determined by a luminometer. Y-axis: % from the Day 1 values (200% means double from the Day 1 ). X-axis: Days after stroke.

[0023] Figure 4A shows CD171 -specificity. Three different control plasma samples (♦•■) and buffer alone (o) were applied to ELISA wells, where anti-CD171 or control IgG were previously immobilized, followed by anti-DD probe reaction. Y axis was ELISA readings of relative light unit (RLU). Figures 4B-4C. NDE-specificity. Three different control plasma samples (♦•■) and buffer alone (o) were first applied to anti-CD171-immoblilized Finlayson Docket No.: 2200-1405

[0024] ELISA wells (Before), then the supernatants were further transferred to the second anti- CD171-immoblilized ELISA wells (After) for the analysis of NDE reduction. Then each well was reacted with anti-CD9 probes for the quantification of NDE (CD171+CD9+double positive signals) (B) or anti-DD probes for the quantification of DD+NDE (CD171+DD+double positive signals) (C). Figure 4D. DD specificity. Three different control plasma samples (♦•■) and buffer alone (o) were applied to anti-CD171-immoblilized ELISA wells, followed by dilution series of anti-DD probes (full strength (f.s.), , and % dilution) to show dose dependent anti-DD reaction (Fig. 4D). Such probe reaction was blocked when DD antigen (0.14 mg / mL) was included in the probe solution (Fig. 4D, left).

[0025] Figure 5A-B shows the quantification of plasma levels of DD+NDE. We first identified high titer plasma of the levels of CD171+DD+(DD+NDE), and assigned it as 100 units / mL (U / mL) as a quantification standard. Figure 5A. Dilution curve of standard plasma. Figure 5B. Results of human samples. We measured DD+NDE in 23 different control plasma and patient samples collected 1 , 3, 5, and 7 days after hemorrhagic stroke. ELISA readings were converted to U / mL using the 4 parameter logistic formula of standard dilution curve.

[0026] Figure 6A-D shows DD+NDE levels gradually increased from Day 1 values (D U / mL, 11 patients) and peaked around 5-7 days in patients with hematoma volumes size less than 5 mL (Figure 6A), whereas patients with ICH volumes >5mL showed no or slow increases (Figure 6B). Such increases were seen in patients with favorable outcome (Figure 6C) versus those with unfavorable outcome (Figure 6D).

[0027] Figure 7 shows plasma levels of oligodendrocyte-derived exosomes by measuring CD9+MOG+double positive signals (CD9 on MOG, Y-axis). Deep intracerebral hemorrhage (hemorrhagic stroke in white matter) (n=7, 2 male and 5 female, 57+7.7 years old). Blood samples were collected 1 , 3, 5, and 7 days after stroke with some exception. Finlayson Docket No.: 2200-1405

[0028] DETAILED DESCRIPTION

[0029] Patterns of release of extracellular vesicles (EV) and their associated proteins into the blood render a window into cellular events occurring in the brain for days following intracerebral hemorrhage (ICH). Brain derived EV quantification offers a novel method of elucidating mechanisms of secondary damage and recovery after ICH. Various plasma or serum proteins have been studied extensively for the assessment of post-hemorrhagic stroke. In this study, we focused on the biomarkers present on the surface of neuron- derived exosomes / EV (NDE)and astrocyte-derived exosomes / EV (ADE) and oligodendrocyte derived exosomes (ODE). Locally released biomolecules in the brain are incorporated or bound to the surface of NDE. Such protein biomarker complexed NDE and ADE are released into bloodstream. Thus, by measuring NDE and ADE or ODE surface biomolecules in blood samples from ICH patients, it is possible to monitor posthemorrhage cellular and molecular cascades quantitatively.

[0030] It is to be understood that contemplated embodiments are not limited to the particular methodologies, protocols, cell lines, assays, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments and is in no way intended to limit the scope of contemplated embodiments as set forth in the appended claims.

[0031] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless context clearly dictates otherwise. Thus, for example, a reference to “a fragment” includes a plurality of such fragments, a reference to an “antibody” is a reference to one or more antibodies and to equivalents thereof known to those skilled in the art, and so forth.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which these contemplated embodiments and disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of contemplated embodiments, the preferred methods, devices, and materials are now described. All publications cited herein are incorporated herein by reference in their Finlayson Docket No.: 2200-1405 entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with contemplated embodiments. Nothing herein is to be construed as an admission that contemplated embodiments and elements of the present disclosure are not entitled to antedate such disclosure by virtue of prior invention.

[0033] The practice of the embodiments and contemplated embodiments of the disclosure will utilize, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Gennaro, A.R., ed. (1990) Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; CoIowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag).

[0034] Specifically, methods of measuring the effects of an intracerebral hemorrhage are disclosed herein and include: acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage, utilizing the at least one blood, serum, or plasma sample to isolate at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least one surface associated biomarker, wherein the at least one surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof, and quantifying the at least one surface biomarker to monitor posthemorrhage cellular and molecular cascades in the patient. Finlayson Docket No.: 2200-1405

[0035] In contemplated methods, the extracellular vesicle comprises exosomes, ectosomes, and apoptotic bodies. In contemplated embodiments, the at least one astrocyte-derived extracellular vesicle comprises excitatory amino acid transporter 1 or 2, CD9, or a combination thereof. Also, the at least one oligodendrocyte-derived extracellular vesicle comprises myelin oligodendrocyte glycoprotein (MOG), CD9, or a combination thereof. In addition, the at least one neuron-derived extracellular vesicle comprises CD9, CD171 , or a combination thereof.

[0036] Also, the at least one surface biomarker and / or at least one surface associated biomarker comprises extracellular vesicle markers, intracellular proteins released from damaged brain cells, neurotrophic factors for the recovery of damaged or stressed cells, proteins responsible for thrombosis and fibrinolysis, inflammatory cytokines, aquaporins and proteins released during brain edema. In some contemplated embodiments, the at least one surface biomarker comprises at least one inflammatory cytokine, at least one D-dimer, GFAP, AQP4, IL1 , or a combination thereof.

[0037] In some contemplated embodiments, the at least one surface biomarker comprises at least one blood coagulation biomarker, at least one fibrinolysis biomarker, at least one glial scr formation biomarker, at least one antioxidant defense system biomarker, at least one cell recovery biomarker, at least one neuroinflammation biomarker, at least one cell death biomarker, at least one brain edema biomarker, or a combination thereof. In these contemplated embodiments, the at least one blood coagulation biomarker comprises coagulation factors (Factor l-XIII), the at least one fibrinolysis biomarker comprises plasmin, plasminogen activators, breakdown products of fibrin, including D-dimer and other Fibrin Degradation Products (FDPs) or a combination thereof, the at least one glial scr formation biomarker comprises Neurocan (NCN), Brevican (BCAN), and Versican (VCAN), Tenascins, Hyaluronic Acid (HA), Fibronectin (FN1 ), Laminin, Collagens, Osteopontin, Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS), Tissue Plasminogen Activator (tPA), Plasmin, Heparanase, Chondroitinase, Cathepsins, and Tissue Inhibitors of Metalloproteinases (TIMPs) or a combination thereof. Finlayson Docket No.: 2200-1405

[0038] In other contemplated embodiments, the at least one antioxidant defense system biomarker comprises superoxide dismutase (SOD3), Glutathione Peroxidase 3 (GPx3), Peroxiredoxin 4 (Prx4), Catalase, Thioredoxin (Trx), Heme Oxygenase-1 (HO-1 ), Paraoxonase 2 (PON2) or combinations thereof, the at least one cell recovery biomarker comprises Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophins, Glial Cell Line-Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Fibroblast Growth Factor (FGF), Insulin-Like Growth Factor-1 (IGF-1 ), Transforming Growth Factor-B (TGFB), Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGE), Neurturin (NRTN), Neuregulin, and Artemin or a combination thereof, and the at least one neuroinflammation biomarker comprises cytokines and chemokines.

[0039] In yet other contemplated embodiments, the at least one cell death biomarker comprises ubiquitin carboxy-terminal hydrolase L1 (UCHL1 ), neurofilaments, synaptic proteins, Microtubule-Associated Proteins (MAPs), Neuronal Nuclei Protein (NeuN), Growth-Associated Protein 43 (GAP-43), Calbindin, Parvalbumin, Neurogranin, 15 Postsynaptic Density Protein 95 (PSD-95), Drebrin, a-Synuclein (SNCA), TAR DNABinding Protein 43 (TDP-43), Neuron-Specific Enolase (NSE), glial fibrillary acidic protein (GFAP), S100 Calcium Binding Proteins (SWOB), Glutamine Synthetase (GS), Vimentin, and Astrocytic Phosphoprotein (AP) or combinations thereof, and the at least one brain edema biomarker comprises aquaporin 4 (AQP4), Albumin, Immunoglobulins, Fibrinogen, a1 -Antitrypsin, a2-Macroglobulin, Transferrin, Haptoglobin, Ceruloplasmin, C-reactive Protein (CRP), Complement Proteins, Lipoproteins, Clotting Factors, Betaglobulins, Gamma-globulins, Myoglobin, Hypoxia-inducible factor 1 -alpha (HIF-1 a), vascular endothelial growth factor (VEGF) or combinations thereof.

[0040] In contemplated methods, acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage includes acquiring the at least one blood, serum, or plasma sample from the brain of a patient. In addition, contemplated methods are also a measure of the status of post-intracranial hemorrhage cellular stress, death, and recovery, thrombosis and fibrinolysis, hemorrhage expansion and brain Finlayson Docket No.: 2200-1405 edema, inflammation, or a combination thereof. In some contemplated embodiments, the intracranial hemorrhage is atraumatic.

[0041] In addition, methods of measuring the effects of brain trauma and degree of recovery are also disclosed herein and include: acquiring at least one blood, serum, or plasma sample from a patient suffering from a brain trauma, neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte- derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least one surface associated biomarker, wherein the at least one surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof, and quantifying the at least one surface biomarker to monitor a cellular cascade and a molecular cascade in the patient.

[0042] Additional contemplated methods of measuring the effects of an intracerebral hemorrhage also include: acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage, utilizing the at least one blood, serum, or plasma sample to isolate at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least a first surface associated biomarker and at least a second biomarker, wherein the first surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof and wherein the second surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof and is also a coagulation biomarker, a fibrinolysis biomarker, an inflammatory biomarker, or a combination thereof, and quantifying the first surface biomarker and the second biomarker to monitor post-hemorrhage cellular and molecular cascades in the patient. Finlayson Docket No.: 2200-1405

[0043] It should be understood that the terms “exosome” and “EV” are used interchangeably herein. Exosomes range in size from 30 to 150 nanometers, are membrane-bound extracellular vesicles (EVs) that are produced in the endosomal compartment of most eukaryotic cells. In multicellular organisms, exosomes and other EVs are found in biological fluids including saliva, blood, urine and cerebrospinal fluid. EVs have specialized functions in physiological processes, from coagulation and waste management to intercellular communication.

[0044] In some contemplated embodiments, contemplated biomarkers comprise a neuron-specific protein (e.g., synaptosome associated protein 25 (SNAP25), neurogranin (NRGN), tau, phosphorylated tau, a£-42, ap-40, along with aggregated forms, and synaptophysin), an astrocyte-specific protein (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 or 2 (EAAT1 or EAAT2), a microglia-specific protein (CD11 b), an oligodendrocyte-specific protein (e.g., myelin basic protein (MBP), an oligodendrocyte myelin glycoprotein (OMG), a cytosolic protein (e.g., glyceraldehyde- 3-phosphate dehydrogenase (GAPDH), alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1 , REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1 , GSK, PLAP, CSH1 , PSG1 , or FasL), a chemokine (CX3CL1 , CCLs, CXCLs), UCL, or cytokine (interleukins, such as IL1 b, IL6, IL34, IL12B or FasL).

[0045] Biological Sample

[0046] A biological sample comprising vesicles (e.g., exosomes) may be obtained from a subject. The biological sample obtained from the subject is typically blood, but can be any sample from bodily fluids, tissue or cells comprising the vesicles to be analyzed. The biological sample may include, but is not limited to, whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cerebrospinal fluid, a cervical swab, tears, saliva, a buccal swab, skin, organs, and biopsies. Alternatively, exosomes can be obtained from cultured cells by collection of secreted exosomes from the surrounding culture media. Finlayson Docket No.: 2200-1405

[0047] In some embodiments, the biological sample of the contemplated subject matter is obtained from blood, plasma, or serum. In some embodiments, about 1 -10 mL of blood is drawn from a subject. In other embodiments, about 10 -50 mL of blood is drawn from a subject. Blood can be drawn from any suitable area of the body, including an arm, a leg, or blood accessible through a central venous catheter. In some embodiments, blood is collected following a treatment or activity. For example, blood can be collected following a medical exam. The timing of collection can also be coordinated to increase the number and / or composition of vesicles (e.g., exosomes) present in the sample. For example, blood can be collected following exercise or a treatment that induces vascular dilation.

[0048] Blood may be combined with various components following collection to preserve or prepare samples for subsequent techniques. For example, in some embodiments, blood is treated with an anticoagulant, a cell fixative, a protease inhibitor, a phosphatase inhibitor, a protein, a DNA, or an RNA preservative following collection. In some embodiments, blood is collected via venipuncture using vacuum collection tubes containing an anticoagulant such as EDTA or heparin. Blood can also be collected using a heparin-coated syringe and hypodermic needle. Blood can also be combined with components that will be useful for cell culture. For example, in some embodiments, blood is combined with cell culture media or supplemented cell culture media (e.g., cytokines).

[0049] Enrichment or Isolation of Vesicles (Exosomes, Microparticles, Microvesicles, Nanosomes, Extracellular Vesicles, and Ectosomes)

[0050] Samples can be enriched for vesicles through positive selection, negative selection, or a combination of positive and negative selection. In some embodiments, vesicles are directly captured. In other embodiments, blood cells are captured and vesicles are collected from the remaining biological samples. In some embodiments, the vesicles enriched in the biological samples are exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, or ectosomes. In some embodiments, the vesicles enriched in the biological samples are neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes. Finlayson Docket No.: 2200-1405

[0051] Samples can also be enriched for vesicles based on differences in the biochemical properties of vesicles. For example, samples can be enriched for vesicles based on antigen, nucleic acid, metabolic, gene expression, or epigenetic differences. In some of the embodiments based on antigen differences, antibody-conjugated magnetic or paramagnetic beads in magnetic field gradients or fluorescently labeled antibodies with flow cytometry are used. In some of the embodiments based on nucleic acid differences, flow cytometry is used. In some of the embodiments based on metabolic differences, dye uptake / exclusion measured by flow cytometry or another sorting technology is used. In some of the embodiments based on gene expression, cell culture with cytokines is used. Samples can also be enriched for vesicles based on other biochemical properties known in the art. For example, samples can be enriched for vesicles based on pH or motility. Further, in some embodiments, more than one method is used to enrich for vesicles. In other embodiments, samples are enriched for vesicles using antibodies, ligands, or soluble receptors.

[0052] In other embodiments, surface markers are used to positively enrich vesicles in the sample. In some embodiments, the vesicles are exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, or ectosomes. In other embodiments, NCAM, CD171 , CD9, CD63, CD81 , SNAP25, EAAT1 , EAAT2, OMG, MOG, neuronspecific enolase, diverse neuron or astrocyte adhesive proteins, microglial CD18 / 11 , or CD3 T cell membrane cell surface markers are used to enrich for exosomes. In some embodiments, cell surface markers that are not found on vesicles populations are used to negatively enrich vesicles by depleting cell populations. Flow cytometry sorting may also be used to further enrich for exosomes using cell surface markers or intracellular or extracellular markers conjugated to fluorescent labels. Intracellular and extracellular markers may include nuclear stains or antibodies against intracellular or extracellular proteins preferentially expressed in vesicles. Cell surface markers may include antibodies against cell surface antigens that are preferentially expressed on exosomes (e.g., NCAM). In some embodiments, the cell surface marker is a neuron-derived exosome surface marker, including, for example, NCAM or CD171. In some embodiments, a monoclonal NCAM, CD9, CD63, CD81 , neuron-specific enolase or Finlayson Docket No.: 2200-1405

[0053] CD171 antibody is used to enrich or isolate exosomes from the sample. In certain aspects, the NCAM, CD9, CD63, CD81 , neuron-specific enolase or CD171 antibody is biotinylated. In this embodiment, biotinylated NCAM or CD171 antibody can form an antibody-exosome complex that can be subsequently isolated using streptavidin-agarose resin or beads. In other embodiments, the NCAM, CD9, CD63, CD81 , neuron-specific enolase or CD171 antibody is a monoclonal anti-human NCAM, CD9, CD63, CD81 , neuron-specific enolase or CD171 antibody. In other embodiments, the cell surface marker is a neuron-specific protein (e.g., synaptosome associated protein 25 (SNAP25), neurogranin (NRGN), tau, phosphorylated tau, a0-42, and synaptophysin), an astrocytespecific protein (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1 )), a microglia-specific protein (CD11 b), an oligodendrocyte-specific protein (e.g., myelin basic protein (MBP), an oligodendrocyte myelin glycoprotein (OMG), a cytosolic protein (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha- synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1 , REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1 , GSK, PLAP, CSH1 , PSG1 , or FasL), or a chemokine (CX3CL1 ), UCL, or cytokine (IL1 b, IL6, IL34, FasL, or IL12B).

[0054] In some embodiments, enriched vesicles from the biological sample are subsequently enriched for a specific type of vesicle. For example, the biological sample is enriched for exosomes and then the enriched exosomes are subsequently enriched for neural-derived exosomes. In some embodiments, the biological sample is enriched for individual neural cell sources of vesicles. In certain aspects, the neural cell sources of vesicles are microglia, neurons, or astrocytes.

[0055] In other embodiments, vesicles are isolated or enriched from a biological sample by a method comprising: contacting a biological sample with an agent under conditions wherein a vesicle present in said biological sample binds to said agent to form a vesicleagent complex; and isolating said vesicle from said vesicle-agent complex to obtain a sample containing said vesicle, wherein the purity of vesicles present in said sample is greater than the purity of vesicles present in said biological sample. In certain Finlayson Docket No.: 2200-1405 embodiments, the agent is an antibody or a lectin. Lectins useful for forming a vesicle- lectin complex are described in U.S. Patent Application Publication No. 2012 / 0077263.

[0056] In some embodiments, the vesicle is an exosome, a microparticle, a microvesicle, nanosomes, extracellular vesicles, or an ectosome. In some embodiments, the exosomes are neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes. In some embodiments, multiple isolating or enriching steps are performed. In certain aspects of the present embodiment, a first isolating step is performed to isolate exosomes from a blood sample and a second isolating step is performed to isolate neural-derived exosomes from other exosomes.

[0057] In yet other embodiments, the methods further comprise releasing the vesicle from the vesicle-agent complex. In other embodiments, the vesicle is released by exposing the vesicle-agent complex to low pH between 3.5 and 1 .5. In other embodiments, the vesicle is released using a competing peptide that competes for the binding of the selection antibody used in the methods of the present contemplated subject matter. In yet other embodiments, the released vesicle is neutralized by adding a high pH solution. In other embodiments, the released vesicle is lysed by incubating the released vesicles with a lysis solution. In still other embodiments, the lysis solution contains inhibitors for proteases and phosphatases. It should be understood that there are contemplated embodiments where lysis of exosomes is not necessary or needed in order to carry out or implement contemplated embodiments of the present disclosure. It should also be understood that intact exosomes may be bound to a solid matrix and subsequently analyzed for EV associated surface biomarkers.

[0058] Biomarkers

[0059] Biomarker levels on vesicles are assayed in a biological sample obtained from a subject having or at-risk of having a disease. In some embodiments, biomarker levels on vesicles are assayed in a biological sample obtained from a subject having or at-risk of having asymptomatic brain trauma and / or intracerebral hemorrhage. In some embodiments, one or more biomarkers are selected from the group consisting of a Finlayson Docket No.: 2200-1405 neuron-specific protein (e.g., synaptosome associated protein 25 (SNAP25), neurogranin (NRGN), tau, and synaptophysin), an astrocyte-specific protein (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1 )), a microglia-specific protein (CD11 b), an oligodendrocyte-specific protein (e.g., myelin basic protein (MBP), an oligodendrocyte myelin glycoprotein (OMG), MOG, and an extracellular vesiclespecific protein (dopamine transporter, DAT). In another embodiment, the biomarkers are CD171 , phosphorylated tau T181 , SNCA, and NRGN. In other embodiments, the biomarkers are acetylcholinesterase (AchE), Lysosomal Associated Membrane Protein 1 (LAMP1 ), CTSD, RE1 Silencing Transcription Factor (REST), synaptotagmin (SYT), monocyte chemotactic protein-^ (CCL2), IL34, glycogen synthase (GYS), (OR), death receptor 6 (DR6), heat shock protein (HSP), IL12beta, alpha-beta (A0), and beta- secretase (BACE). In some embodiments, one or more biomarkers are selected from the group consisting of cytosolic proteins, secretory proteins, membrane proteins and receptors and their pathological forms, including aggregates and mutated ones. Biomarkers of the present contemplated subject matter include neurotransmitter receptors, such as, for example, dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1 -6, 5. B1 , B2), and glutamate receptors (1 and 2). Other receptor biomarkers of the present contemplated subject matter include, insulin receptors, tumor necrosis factor receptors superfamily (TRAL, TNF receptor, death receptor 5 and 6), and neuropeptide receptors (orexin receptor, opioid receptor KOR). Biomarkers of the present contemplated subject matter include membrane proteins, such as, for example, EpCAM, PD-L1 , ErbB2, CK19, TOR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. Other known neurological disorder biomarkers may be used in combination with the biomarkers of the present contemplated subject matter. Examples of such biomarkers are provided in US Patent Application Pub. No. 2015 / 0119278, the contents of which are hereby incorporated by reference.

[0060] One of ordinary skill in the art has several methods and devices available for the detection and analysis of the markers of the instant disclosure. With regard to polypeptides or proteins on vesicles in patient test samples, immunoassay devices and methods are often used. These devices and methods can utilize labeled molecules in Finlayson Docket No.: 2200-1405 various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amounts of analytes without the need for a labeled molecule.

[0061] Preferably the markers are analyzed using an immunoassay, although other methods are well known to those skilled in the art (for example, the measurement of marker RNA levels). The presence or amount of a marker is generally determined using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay may be utilized, for example, an enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay (IFA), immune-polymerase chain reaction assay, electro-chemiluminescence immunoassay (ECLIA), radioimmunoassay (RIA), competitive binding assay, planar waveguide technology, and the like. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.

[0062] The use of immobilized antibodies specific for the surface markers on vesicles is also contemplated by the present contemplated subject matter. The antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper), and the like. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample to capture vesicles through binding to surface markers and then processed quickly through washes and detection steps with detection reagents, as described above, to generate a measurable signal, such as a colored spot.

[0063] The analysis of a plurality of markers may be carried out separately or simultaneously with one test sample. Several markers on vesicles may be captured and / or detected using a combination of multiple capture agents and / or detection agents Finlayson Docket No.: 2200-1405 in one test for efficient processing of multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same individual. Such testing of serial samples will allow the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, would provide useful information about the disease status that includes, but is not limited to identifying the approximate time from onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, identification of the severity of the event, identification of the disease severity, and identification of the patient's outcome, including risk of future events.

[0064] An assay consisting of a combination of the markers referenced in the instant contemplated subject matter may be constructed to provide relevant information related to differential diagnosis. Such a panel may be constructed using 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers. The analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out methods described within the contemplated subject matter to optimize clinical sensitivity or specificity in various clinical settings.

[0065] The analysis of markers could be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different analytes. Such formats include protein microarrays, or “protein chips” and capillary devices.

[0066] Biomarkers of the present disclosure serve an important role in the early detection and monitoring of asymptomatic brain trauma and / or intracerebral hemorrhage. Markers of such disorders are typically substances found in a bodily sample that can be measured. The measured amount can correlate to underlying disorder or disease pathophysiology, Finlayson Docket No.: 2200-1405 presence or absence of a neurological disorder, probability of a neurological disorder in the future. In patients receiving treatment for their condition the measured amount will also correlate with responsiveness to therapy. In some embodiments, a decrease or increase in the level of one or more biomarkers of the present contemplated subject matter is indicative of a neurological disorder.

[0067] In some embodiments, a biomarker is measured by a method selected from the group consisting of immunohistochemistry, immunocytochemistry, immunofluorescence, immunoprecipitation, electro-chemiluminescence immunoassay, radioimmunoassay, immune-polymerase chain reaction, western blotting, and ELISA.

[0068] Clinical Assay Performance

[0069] The methods of the present contemplated subject matter may be used in clinical assays to diagnose or prognose asymptomatic brain trauma and / or intracerebral hemorrhage in a subject, identify a subject at risk of asymptomatic brain trauma and / or intracerebral hemorrhage, and / or for prescribing a therapeutic regimen or predicting benefit from therapy in a subject having asymptomatic brain trauma and / or intracerebral hemorrhage. Clinical assay performance can be assessed by determining the assay’s sensitivity, specificity, area under the ROC curve (AUC), accuracy, positive predictive value (PPV), and negative predictive value (NPV). Disclosed herein are assays for diagnosing or prognosing asymptomatic brain trauma and / or intracerebral hemorrhage in a subject, identifying a subject at risk of asymptomatic brain trauma and / or intracerebral hemorrhage, or for prescribing a therapeutic regimen or predicting benefit from therapy in a subject having asymptomatic brain trauma and / or intracerebral hemorrhage.

[0070] The clinical performance of the assay may be based on sensitivity. The sensitivity of an assay of the present contemplated subject matter may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. The clinical performance of the assay may be based on specificity. The specificity of an assay of the present contemplated subject matter may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. The clinical performance of the Finlayson Docket No.: 2200-1405 assay may be based on area under the ROC curve (AUC). The AUC of an assay of the contemplated subject matter may be at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. The clinical performance of the assay may be based on accuracy. The accuracy of an assay of the contemplated subject matter may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.

[0071] Compositions

[0072] Compositions useful in the methods of the present subject matter include compositions that specifically recognize a biomarker associated with intercerebral hemorrhage. Such compositions may include capture agents and / or detection agents that recognize, for example, a neuron-specific protein biomarker, such as synaptosome associated protein 25 (SNAP25), 00-42, neurogranin (NRGN), tau, phosphorylated tau, and synaptophysin, an astrocyte-specific protein biomarker, such as glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 or 2 (EAAT1 or EAAT2), an oligodendrocyte-specific protein biomarker, such as myelin basic protein (MBP) and oligodendrocyte myelin glycoprotein (OMG), myelin oligodendrocyte glycoprotein (MOG), a microglia-specific protein (CD11 b), a cytosolic protein (e.g., glyceraldehyde-3- phosphate dehydrogenase (GAPDH), alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1 , REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1 , GSK, PLAP, CSH1 , PSG1 , or FasL), or a chemokine (CX3CL1 ), UCL, D-dimer, or cytokine (IL1b, IL6, IL34, FasL, or IL12B).

[0073] In yet other embodiments, the composition is selected from the group consisting of a peptide, a nucleic acid, an antibody, and a small molecule.

[0074] In certain embodiments, the contemplated subject matter relates to compositions that specifically detect a biomarker associated with intercerebral hemorrhage. As detailed elsewhere herein, the contemplated subject matter is based upon the finding that GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated Tau, synaptophysin, 00-42, CX3CL1 , IL1 b, IL34, CD81 , CD63, CD171 , SNAP25, EAAT1 , SNCA, CD11 b, OMG, MOG, AchE, LAMP1 , REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, Finlayson Docket No.: 2200-1405

[0075] OR, DR6, HSP, IL12b, A0, and BACE can be used as biomarkers for AD and other neurological disorders. In some embodiments, the compositions of the contemplated subject matter specifically bind to and detect such biomarkers. For example, a composition may comprise a solid support comprising capture agents associated therewith that selectively bind to CD81 , CD63, CD171 , SNAP25, EAAT1 , CD11b, OMG, or MOG. In another example, a composition may comprise detection agents that selectively bind to GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated Tau, synaptophysin, ap-42, SNCA, CX3CL1 , IL1 b, IL34, OMG, AchE, LAMP1 , REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Ap, and / or BACE.

[0076] In some embodiments, the composition comprises an antibody, where the antibody specifically binds to a biomarker or vesicles of the contemplated subject matter. The term “antibody” as used herein and further discussed below is intended to include fragments thereof which are also specifically reactive with a biomarker or vesicle (e.g., exosome). Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. Antigenbinding portions may also be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, singlechain antibodies (scFv), single domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. In certain embodiments, the antibody further comprises a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor).

[0077] In certain embodiments, an antibody of the present disclosure is a monoclonal antibody, and in certain embodiments, the contemplated subject matter makes available methods for generating novel antibodies that specifically bind the biomarker or the Finlayson Docket No.: 2200-1405 exosome of the contemplated subject matter. For example, a method for generating a monoclonal antibody that specifically binds a biomarker or exosome, may comprise administering to a mouse an amount of an immunogenic composition comprising the biomarker or exosome, or fragment thereof, effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monoclonal antibody that binds specifically to the biomarker or exosome. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the biomarker or exosome. The monoclonal antibody may be purified from the cell culture.

[0078] The term “specifically reactive with” as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g., a biomarker or exosome) and other antigens that are not of interest. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross-reacting polypeptides. One characteristic that influences the specificity of an antibody: antigen interaction is the affinity of the antibody for the antigen. Although the desired specificity may be reached with a range of different affinities, generally preferred antibodies will have an affinity (a dissociation constant) of about 10-6, 10-7, 108, 10-9or less.

[0079] Antibodies can be generated to bind specifically to an epitope of an exosome or a biomarker of the present disclosure, including, for example, neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes, or neuron-specific proteins selected from the group consisting of synaptosome associated protein 25 (SNAP25), neurogranin (NRGN), tau, phosphorylated tar, and synaptophysin, astrocyte-specific proteins selected from the group consisting of glial fibrillary acidic protein (GFAP) and excitatory amino acid Finlayson Docket No.: 2200-1405 transporter 1 or 2 (EAAT1 or EAAT2), and oligodendrocyte-specific proteins selected from the group consisting of myelin basic protein (MBP) and oligodendrocyte myelin glycoprotein (OMG), MOG, a microglia-specific protein (CD11 b), and chemokine (CX3CL1 ) or cytokine (IL1 b, IL34, FasL, or IL12B). In another embodiment, the antibody generated is an anti-CD171 antibody, an anti-synaptosome associated protein 25 (SNAP25) antibody, an anti-neurogranin (NRGN) antibody, an anti-tau antibody, an anti-synaptophysin antibody, and anti-CD63 antibody, an anti-ap-42 antibody, an anti- CD81 antibody, an anti-CTD antibody, an anti-GAPDH antibody, an anti-IL1 b antibody, an anti-IL34 antibody, an anti-CX3CL1 antibody, an anti-glial fibrillary acidic protein (GFAP) antibody, an anti-excitatory amino acid transporter 1 or 2 (EAAT1 or EAAT2) antibody, an anti-SNCA antibody, an anti-TH antibody, and anti-CD11 b antibody, an anti-myelin basic protein (MBP) antibody, an anti-oligodendrocyte myelin glycoprotein (OMG) antibody, an anti-myelin oligodendrocyte glycoprotein (MOG) antibody, an anti-dopamine transporter (DAT) antibody, an anti-AchE antibody AchE, an anti-LAMP1 antibody LAMP1 , an anti-REST antibody REST, an anti-SYT antibody SYT, an anti-SYP antibody, an anti-SYNPO antibody, an anti- PSD95 antibody, an anti-SV2A antibody, an anti-CCL2 antibody CCL2, an anti-l L34 antibody IL34, an anti-GYS antibody GYS, an anti-OR antibody OR, an anti-DR6 antibody DR6, an anti-HSP antibody HSP, an anti- IL12b antibody IL12b, an anti-Ap antibody Ap, or an anti-BACE antibody BACE.

[0080] In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. A variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Md.), western blots, immunoprecipitation assays, immunocytochemistry, and immunohistochemistry.

[0081] In some embodiments, the present disclosure relates to compositions used for identifying or treating intercerebral hemorrhage. As detailed elsewhere herein, the present disclosure is based upon the findings that the levels of CD81 , GAPDH, CTSD, Finlayson Docket No.: 2200-1405

[0082] NRGN, MBP, GFAP, Tau, phosphorylated Tau (e.g., T181 ), synaptophysin, CD63, a0- 42, SNCA, CX3CL1 , IL1 b, IL34, AchE, LAMP1 , REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, A , and / or BACE are implicated in the pathology of asymptomatic brain trauma and / or intracerebral hemorrhage.

[0083] In some embodiments, biomarkers inside vesicles are analyzed in addition to the surface biomarkers. In certain embodiments, the present disclosure relates to exosome surface associated biomarkers in biological samples obtained from a subject.

[0084] Contemplated target biomarkers are found in Figure 1 and disclosed herein. Contemplated methods of monitoring hemorrhage are disclosed herein that include: a) providing a biological sample; and b) detecting double positive signals of both exosome or extracellular vesicle markers and coagulation or fibrinolysis markers.

[0085] The following conditions and processes are relevant with respect to contemplated biomarkers.

[0086] Blood Coagulation. After the bleeding in the brain parenchyma, blood is coagulated with serial activation of coagulation factors (Factor l-XIII). These factors are released in the lesion.

[0087] Fibrinolysis. This is the process by which the body breaks down blood clots. Fibrinolysis involves a cascade of enzyme (Plasmin) and cofactors (Plasminogen Activators) to release breakdown products of fibrin, including D-dimer and other Fibrin Degradation Products (FDPs).

[0088] Glial scar formation. After bleeding, damaged lesions must be concealed to prevent further damages. This is so called glial scar formation, which involves the accumulation of several extracellular matrix (ECM) proteins. Key ECM proteins are Chondroitin Sulfate Proteoglycans (CSPGs), including Neurocan (NCN), Brevican (BCAN), and Versican (VCAN). Other ECM proteins are Tenascins, Hyaluronic Acid (HA), Fibronectin (FN1 ), Finlayson Docket No.: 2200-1405

[0089] Laminin, Collagens, and Osteopontin. Several enzymes degrade or remodel the extracellular matrix (ECM) to regulate the scar formation process. These enzymes are Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS), Tissue Plasminogen Activator (tPA), Plasmin, Heparanase, Chondroitinase, Cathepsins, and Tissue Inhibitors of Metalloproteinases (TIMPs).

[0090] Antioxidant defense system. Stressed neuronal cells release various enzymes as an antioxidant defense system which includes extracellular superoxide dismutase (SOD3), Glutathione Peroxidase 3 (GPx3), Peroxiredoxin 4 (Prx4), Catalase, Thioredoxin (Trx), Heme Oxygenase-1 (HO-1 ), Paraoxonase 2 (PON2).

[0091] Cell recovery. Stressed neuronal cells and astrocytes also release various neurotrophic factors to initiate recovery process. These factors include Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophins, Glial Cell Line-Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Fibroblast Growth Factor (FGF), Insulin-Like Growth Factor-1 (JGF-1), Transforming Growth Factor-p (TGF- P), Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Neurturin (NRTN), Neuregulin, and Artemin.

[0092] Neuroinflammation. Severe damage induces neuroinflammation by secreting various cytokines and chemokines, including IL1 .

[0093] Cell death. When neuron is severely damaged, dying neuronal cells release intracellular proteins, such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1 ), neurofilaments, synaptic proteins, Microtubule-Associated Proteins (MAPs), Neuronal Nuclei Protein (NeuN), Growth-Associated Protein 43 (GAP-43), Calbindin, Parvalbumin, Neurogranin, Finlayson Docket No.: 2200-1405

[0094] Postsynaptic Density Protein 95 (PSD-95), Drebrin, a-Synuclein (SNCA), TAR DNA- Binding Protein 43 (TDP-43), Neuron-Specific Enolase (NSE), etc. When astrocytes are severely damaged, dying astrocytes release astrocyte-specific intracellular proteins to extracellular space. These include glial fibrillary acidic protein (GFAP), S100 Calcium- Binding Proteins (SWOB), Glutamine Synthetase (GS), Vimentin, and Astrocytic Phosphoprotein (AP).

[0095] Brain edema. Astrocytes are responsible for water content in the brain via water channel protein, aquaporin 4 (AQP4) to control brain edema. In hemorrhagic stroke, extracellular (vasogenic) or intracellular (cytotoxic) brain edema are induced. Vasogenic edema is an excess fluid (plasma) in the brain, meaning that various plasma proteins are present in the parenchyma of the brain. Such proteins are Albumin, Immunoglobulins, Fibrinogen, a1 -Antitrypsin, a2 -Macroglobulin, Transferrin, Haptoglobin, Ceruloplasmin, C-reactive Protein (CRP), Complement Proteins, Lipoproteins, Clotting Factors, Beta-globulins, Gamma-globulins, and Myoglobin. Cytotoxic edema is induced by hypoxia of the cells. Hypoxia-inducible factor 1 -alpha (HIF-1a) is upregulated in response to low oxygen levels, subsequently induces the expression of vascular endothelial growth factor (VEGF).

[0096] AQP4 is a transmembrane protein expressed on the surface of astrocytes and astrocyte-derived exosomes (ADE). Therefore, AQP4 are biomarkers of ADE and all others are biomarkers of NDE, ADE or oligodendrocyte-derived exosomes (ODE).

[0097] EXAMPLES

[0098] Example 1 : Plasma Sample Study

[0099] In order to demonstrate the efficacy of contemplated embodiments, plasma samples were applied to enzyme-linked immunosorbent assay (ELISA) wells, where neuron-specific anti-CD171 antibodies (ProSci, Poway, CA) or astrocyte specific EAAT1 Antibodies (Bioss, Woburn, MA) were previously immobilized. After capturing and Finlayson Docket No.: 2200-1405 isolating CD171+NDE or EAAT1 ADE in each ELISA well, various probes were applied as a sandwich chemiluminescent ELISA by maintaining NDE intact, not lysed or permeabilized. Resultant ELISA readings were relative light units (RLU) in a luminometer. Biomarker+CD171+or EAAT1 double positive signals indicate the quantity of each biomarker per NDE or ADE respectively.

[0100] In our initial study over the 8 days following ICH, NDE (CD9 / CD171 ) and ADE (EAAT1 / CD9) levels trended upward. IL-1 B (BioLegend, San Diego, CA) / NDE levels trended downward and UCHL-1 (Novus, Centennial, CO) / NDE trends were mixed. GFAP (R&D Systems, Minneapolis, MN) / ADE and AQP4 (Abbexa, Cambridge, UK) / ADE trended toward an overall decrease, while demonstrating a bimodal response at days 3- 4 and 6-7 in several cases, as shown in Figures 2 and 3. Exosome responses were not correlated with ICH volume.

[0101] Specifically, Figure 2 shows plasma samples that were applied to anti-CD171 - immobilized ELISA wells to capture NDE. After removing unbound materials from ELISA wells, wells were reacted with biotinylated antibodies against CD9 (Fig. 2 left) for the quantification of captured NDE, ILI beta (Fig. 2, 2ndfrom left) for the analysis of neuroinflammation, BDNF (Fig. 2, 2ndfrom right) for the analysis of brain’s recovery function, and UCHL-1 (Fig. 2, right) for the analysis of neuronal cell death, respectively. Biotinylated antibodies were further reacted with streptavidin-horseradish peroxidase, followed by chemiluminescent substrate, SuperSignal, and relative light units (RLU) were determined by a luminometer. Y-axis: % from the Day 1 values (200% means double from the Day 1 ). X-axis: Days after stroke.

[0102] In addition, Figure 3 shows plasma samples that were applied to anti-EAAT1 - immobilized ELISA wells to capture ADE. After removing unbound materials from ELISA wells, wells were reacted with biotinylated antibodies against CD9 (Fig. 3 left) for the quantification of captured ADE, GFAP (Fig. 3, middle) for the analysis of astrocyte conditions, and AQP4 (Fig. 3, right) for the analysis of brain edema, respectively. Biotinylated antibodies were further reacted with streptavidin-horseradish peroxidase, followed by chemiluminescent substrate, SuperSignal, and relative light units (RLU) were Finlayson Docket No.: 2200-1405 determined by a luminometer. Y-axis: % from the Day 1 values (200% means double from the Day 1 ). X-axis: Days after stroke.

[0103] Subsequent expanded studies on NDE expression of additional markers were conducted to include D-dimer (Abbexa), NCAN (Biorbyt, Durham, NC) MMP2 (MyBiosourece, San Diego, CA), SOD3 (MyBiosource), VEGF (Biorbyt), IL18 (RayBiotech, Peachtree Corners, GA), Albumin (Bioss) and TNF alpha (Biorbyt). Levels of all but D-dimer were found to be below the level of detection.

[0104] In another study, serial plasma samples (Day 1 -Day7) from 11 hemorrhagic stroke patients (#22-#113) and 8 healthy control adults were used to measure D-dimer on NDE.

[0105] The ELISA results (RLU) of D-dimer are shown in Figure 4, where Day 1 (D1 ) patients’ samples were not different from control subjects, whereas at Day 3 (D3), values were significantly higher (p=0.004) than those of control. This time course is reasonable.

[0106] Although numerous reports have shown plasma D-dimer analysis, no prior art for D-dimer on NDE has been demonstrated. D-dimer is a marker for ischemic stroke, because blood coagulation and fibrinolysis are induced inside blood vessels. However, in hemorrhagic stroke, blood coagulation and fibrinolysis are induced outside of blood vessels. Thus, plasma D-dimer measurement alone is considered as meaningless. We found that D-dimer on NDE is a useful and specific indicator of hemorrhagic stroke. By monitoring the changes of D-dimer on NDE, physicians can assess the blood coagulation / fibrinolysis process in the brain in each patient, which may then predict outcome and develop personalized interventions.

[0107] In this these studies of contemplated embodiments, we found trends to increased NDE and ADE levels following ICH suggesting early and sustained blood brain barrier breakdown, an early peak and gradual decline in inflammation (IL1 / NDE), early alteration / death of astrocytes (GFAP / ADE) and in many cases, bimodal peaks in cerebral edema (AQP4 / ADE). The trends and significant changes seen in NDE / IL1 , NDE / D-dimer ADE / GFAP and ADE / AQP4 represent novel, unique, and non-obvious brain selective biomarkers that may be indicative of the status and progression of ICH recovery and the brain tissue response post ICH. These unique complexed markers and changes post ICH Finlayson Docket No.: 2200-1405 point to the utility of these assays in ICH and the potential to correlate and monitor results in clinical subgroups based on hemorrhage type, early infection, seizure, midline shift, perihematomal edema, and modified Rankin scale (mRS) and other means of assessment of ICH prognosis and recovery.

[0108] Example 2: Exosome-based assessment of temporal patterns in interleukin-1 beta and aquaporin-4 expression after intracerebral hemorrhage

[0109] Materials and Methods

[0110] Antibodies. Mouse monoclonal antibody against human CD171 (Thermo Fisher Scientific, Waltham, MA), EAAT1 (Abeam, Cambridge, UK), MOG (Thermo Fisher), and CD9 (BD Biosciences, San Jose, CA) were utilized as indicators of neuron derived exosomes (NDE), astrocyte derived exosomes (ADE), and a common exosome membrane marker, respectively. Control mouse lgG2a was obtained from BioLegend (San Diego, CA), and normal mouse IgG was obtained from Equitech-Bio (Kerrville, TX). Interleukin 1 Beta (IL1 P), and aquaphorin-4 (AQP4) used as probes for NDE or ADE associated proteins respectively were obtained from BioLegend, San Diego, CA. All antibodies were commercially sourced and underwent appropriate validation through Western blot, immunohistochemistry, flow cytometry, and / or enzyme-linked immunosorbent assay (ELISA) per the manufacturer. Antibody probes used to target NDE or ADE associated protein were biotinylated for use in a luminescent ELISA. Biotinylation of antibodies was performed using EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific), followed by a spin column procedure to remove unbound biotin

[0111] Antibody immobilization. Antibodies used for immobilization (anti-CD171 , anti-EAAT1 , and control mouse lgG2a and purified normal mouse IgG were diluted in ELISA coating buffer (BioLegend) in a final concentration of 2.5 pg / mL, and 50 pL was applied to ELISA wells (Corning #3923, Sigma Aldrich, St Louis MO). After 1 hour incubation, each well Finlayson Docket No.: 2200-1405 was washed once with phosphate buffered saline (PBS, Thermo Fisher) and incubated with undiluted blocker casein (Thermo Fisher) for another 1 hour. After each well was washed twice with PBS, ELISA wells were stored in a refrigerator.

[0112] ELISA. The assay platform is a sandwich immunoassay with some improvements. In brief, 40 pL samples were applied to ELISA wells and incubated for 1 hour at room temperature. After the first washing step, each well was reacted with biotinylated probes for CD9 or IL1 (3, or AQP-4. All probes were at 50 ng / mL supplemented with 0.8% bovine serum albumin (BSA, Thermo Fisher), 40 pg / mL mouse IgG (Equitech-Bio) for another 1 hour. In order to maintain captured EV intact, tween-20 was not used at any time during the whole procedure. Biotinylation of probe antibodies was carried out by EZ link Sulfo- NHS-LC-Biotin (Thermo Fisher) followed by the spin column procedure to remove free biotin. After the second washing step, each well was reacted with a 1 / 4,000 dilution of poly-horseradish peroxidase (HRP)-conjugated streptavidin (Thermo Fisher) supplemented with 10% BSA (Equitech-Bio) and 30% blocker casein (Thermo Fisher), and incubation was continued for 20 min. After the third washing step, each well was incubated for 5 min with 0.0006% H2O2 (CVS pharmacy, Irvine, CA) diluted in PBS:water (1 :1 ) solution to remove non-specifically bound HRP conjugates. After aspiration of H2O2, each well was mixed with 1 / 3 dilution of chemiluminescent substrate (Super Signal, Thermo Fisher) for 4 min, then relative light units (RLU) were determined by a luminometer (Active GLO, ANSH Labs, Webster, TX). Using a standard plasma, arbitrarily assigned to 100 units / mL (U / mL). ELISA readings of RLU (Relative Light Units) were converted to U / mL by a 4-parameter logistic formula.

[0113] Assay Reproducibility. Previously studies have demonstrated the consistency and reproducibility of the assay for capture and identification of NDE and ADE from peripheral blood. Studies here demonstrate that following capture of NDE and ADE that associated surface markers on EV can be identified. Figure 2 shows concentration dependent linear association duplicate of IL1 [3 and CD9 with NDE can be demonstrated to be Finlayson Docket No.: 2200-1405 concentration dependent with a linear dose response and reproducible with minimal duplicate variation.

[0114] Source of ICH patient samples.

[0115] ICH patient plasma samples were obtained from patients admitted from January 2009 to December 2015 with primary ICH to Duke University Medical Center. Patients were 18 years of age and above, with acute focal neurological deficits within 24 hours of symptom onset or last known well time and confirmed on imaging to have ICH. Approximately 10mL of whole blood was collected in a lavender top tube at admission, 24, 48, 72 and 96 hours after ICH. Within an hour of collection, plasma samples were centrifuged at 2000xg for ten minutes at 4°C and aliquoted into cryotubes. All samples were aliquoted, banked and stored frozen at -80C at Duke University and shipped under dry ice to NanoSomiX for NDE, ADE and biomarker testing. Clinical Variables to be included in analyses were selected based on published correlations with neurological outcome (mRS) after ICH or as a secondary outcome of interest including race, ethnicity, sex, past medical history collected through medical record abstraction, Glasgow Coma Scale (GCS) and NIH Stroke Scale (NIHSS) score upon admission, systolic blood pressure upon admission, hematoma volume as measured by volumetric calculation on initial diagnostic brain computed tomography, hematoma location (“deep" versus “lobar” as noted by the site investigator and adjudicated at the coordinating center), and the presence of intraventricular hemorrhage (IVH) on initial imaging.

[0116] Results:

[0117] In the first seven days following ICH, IL-1 p / NDE levels trended downward (Figure 3). AQP4 / ADE trended toward an overall decrease, while demonstrating a bimodal increase at days 3-4 and 6-7 in several cases. The overall levels of NDE and ADE both reflected a gradual increase. Exosome responses were not correlated with ICH volume, deep versus lobar ICH, midline shift, the presence or absence of intraventricular hemorrhage, infection, or seizure. Finlayson Docket No.: 2200-1405

[0118] Discussion:

[0119] Our methodology allows serial longitudinal pattern characterization of exosome expression in the initial days following ICH, which, when combined with specific biomarkers representing surrogates of events at a cellular level, have potential for development into clinical applications to assess and predict physiological and pathological responses.

[0120] Although our sample size limits clinical conclusions from our findings, trends in AQP4 / ADE and IL-ip / NDE responses suggest plausible detection of a biological signal. In an animal study of lysolecithin injection in rat brains, found a similarly bimodal peak in AQP4, initially at day 3, corresponding with an early brain inflammation state, followed by a second peak at days 7-20, corresponding with the edema resolution phase. The authors had also tested IL-1 [3 and found elevation as early as post-injection day 1 , elevation through the third day suggesting early inflammation, with a subsequent decline persisting through the resolution phase. The persistent decline in IL-1 f3 / NDE may correspond to an early peak in inflammation immediately after ICH, reflecting an expected pattern of elevation followed by downregulation of pro-inflammatory cytokines in the aftermath of ICH.

[0121] While numerous blood biomarkers have been posited to represent specific pathophysiological processes in ICH, trending biomarker levels is not a standard tool in the routine management of ICH, and the utility of biomarkers that have been identified have not been validated by larger studies. In a landscape in which biomarkers have not yet been adopted in the workflow of management of patients with ICH, opportunities exist to develop more specific tools, in which an exosome-based approach can grant organspecificity and cell-specificity by detecting biomarkers within neuron- or astrocyte-derived exosomes. From a temporal perspective, expansion of this pilot analysis into a larger- scale study could uncover distinct patterns in biomarker expression, whose potential correlates with patient outcomes could allow this approach to become a predictive tool to Finlayson Docket No.: 2200-1405 anticipate time courses of brain inflammation and cerebral edema propagation and resolution in patients with ICH.

[0122] Exosome-based assays of IL-1 (3 and AQP4 offer potential as a novel, organospecific, and cell-specific biomarker detection method. Trending expression of IL- 1 |3 / NDE and AQP4 / ADE on days 1 -7 following ICH can characterize temporal patterns of pathophysiology and physiological responses following ICH and potentially help develop clinical tools predicting the generation and resolution of inflammation and edema.

[0123] Example 3: D-dimer on neuron-derived exosomes - An assessment of clot fibrinolysis after intracerebral hemorrhage

[0124] D-dimer (DD) is a fibrin degradation product generated during the breakdown of crosslinked fibrin, a key structural component of blood clots. Its use in diagnostic evaluation of thrombosis and hypercoagulability has drawn attention in ischemic stroke, where intravascular clot formation occurs. A recent meta-analysis found that fibrinogen and D- dimer may be associated with adverse outcomes in acute ischemic stroke. In contrast, intracerebral hemorrhage (ICH) is characterized by bleeding and clotting in brain parenchyma rather than clot formation within blood vessels. Following ICH, hematoma formation must be cleared via fibrinolytic pathways, during which DD is generated locally at the lesion site. In this setting, DD remains confined to the brain parenchyma and does not readily enter the systemic circulation; thus, conventional serological testing for DD has limited clinical utility in ICH due to lack of specificity. Measurement of neuron-derived exosomes (NDEs) selectively detects changes in brain concentrations and offers possible improvement in specificity and stabilization over serology. As proof of principle, their quantification has been applied in Alzheimer’s Disease and acute ischemic stroke. We hypothesize that DD released in the brain binds to NDEs and is subsequently transported into, and measurable in, the bloodstream. By quantifying DD-positive NDE (DD+NDE) post-ICH, brain-specific clot-fibrinolysis cascades can be monitored in real time through serology. This novel approach may ultimately pave the way for new monitoring strategies. Finlayson Docket No.: 2200-1405

[0125] Materials and Methods

[0126] Study Population

[0127] Blood samples from patients with ICH were obtained from clinical protocols approved by the Duke University Institutional Review Board. Informed consent was obtained from each enrolled subject or their legally authorized representative if patients were unable to consent due to impaired capacity, assessed by an informed consent comprehension questionnaire.

[0128] Participants

[0129] From January 2009 to December 2015, all patients aged 18 years and above presenting to Duke University Hospital with an acute focal neurological deficit and computed tomography (CT)-verified spontaneous, non-traumatic ICH within 24 hours of symptom onset (or last known well-time) were approached for consent. Patients with secondary causes of ICH, pregnant females (as verified by urine pregnancy test), and patients with infratentorial hematoma locations were excluded. Of those approached, 182 patients were enrolled in the single-center parent observational study of ICH, from which a pilot cohort of 11 patients were selected, on the basis of having completed the full 7 days of data points and reflecting a range of variation in hemorrhage size, race, presence of intraventricular hemorrhage, deep and lobar hemorrhage, and benign to adverse outcome, with similar distributions of age and sex. One patient was excluded due to endstage renal disease requiring intermittent hemodialysis, so as to remove the possibility of interference of this on exosome levels. The final pilot cohort included four men and six women, with an age range of 45-67 years (average 57 years), hemorrhage volume ranging from 1 ,9-105.5cc (average 36.8cc), initial systolic blood pressure 150-302mm Hg (average 211.7mm Hg) initial Glasgow Coma Scale 3-14 (average 8), initial NIH Stroke Scale 14-28 (average 22), ICU length of stay 2-28 days (average 15.8 days), and 6 month Modified Rankin Scale 3-6 (average 5.1 ). The pilot cohort included five black patients, four white patients, and one Hispanic patient. Finlayson Docket No.: 2200-1405

[0130] Imaging

[0131] Neuroimaging review by the study neurologist of initial CT scans for each participant confirmed diagnosis of spontaneous ICH. Hematoma volumes on diagnostic CTs were measured using AnalyzePro version 1.0 (AnalyzeDirect, Inc., Stilwell, KS) and confirmed by a blinded study neuroradiologist. Hematoma location was dichotomized into lobar and non-lobar.

[0132] Neurological outcome

[0133] Modified Rankin Scale (mRS) 6 months post-ICH was obtained from follow-up phone interviews with patients and LARs5. Phone interviews were conducted by study staff in a standardized fashion after sufficient training by certified mRS assessors. Unfavorable outcome was defined as mRS >3 at 6 months after ICH.

[0134] Blood sampling

[0135] Each enrolled subject had 10 mL of whole blood collected in a lavender-top tube at enrollment or 24±4 hours, 48+4 hours, 72+4 hours, 96+4 hours, 120+4 hours, and 144+4 hours after symptom onset or their last known normal time via venous puncture or by accessing existing central venous, arterial, or peripheral intravenous catheters. Plasma samples were centrifuged at 2000xg for ten minutes at 4°C, and supernatants were aliquoted into five 1 mL aliquots and stored at a -80°C freezer for subsequent measurement of NDEs.

[0136] Reagents.

[0137] All reagents used in this study were consistent with those detailed in our previous publications, except for monoclonal anti-DD and DD antigen (Abbexa). Biotinylation was Finlayson Docket No.: 2200-1405 performed using EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific), followed by a spin column procedure to remove unbound biotin.

[0138] Assay protocol.

[0139] Previously described protocols involve sandwich chemiluminescent enzyme-linked immunosorbent assay (ELISA) with a combination of capture (neuron-specific anti- CD171 , or control IgG) and detection antibodies (anti-DD and exosome common anti- CD9). Captured exosomes remained intact, without lysis or permeabilization, to preserve native structure and surface-bound DD.

[0140] Results

[0141] Assay validation. DD probe signals were higher in anti-CD171 -immobilized ELISA wells compared with control IgG-immobilized counterparts (Figure 4). When CD171+NDE was reduced from plasma samples (Figure 4B), DD+NDE was also reduced (Figure 4C), indicating that DD+NDE was NDE-dependent. Moreover, DD+NDE reaction was blocked by DD antigen (Figure 4D), verifying DD+NDE was DD-dependent.

[0142] Specifically, Figure 4A shows CD171 -specificity. Three different control plasma samples (♦•■) and buffer alone (o) were applied to ELISA wells, where anti-CD171 or control IgG were previously immobilized, followed by anti-DD probe reaction. Y axis was ELISA readings of relative light unit (RLU). Figures 4B-4C. NDE-specificity. Three different control plasma samples (♦•■) and buffer alone (o) were first applied to anti- CD171 -immoblilized ELISA wells (Before), then the supernatants were further transferred to the second anti-CD171 -immoblilized ELISA wells (After) for the analysis of NDE reduction. Then each well was reacted with anti-CD9 probes for the quantification of NDE (CD171+CD9+double positive signals) (B) or anti-DD probes for the quantification of DD+NDE (CD171+DD+double positive signals) (C). Figure 4D. DD specificity. Three different control plasma samples (♦•■) and buffer alone (o) were applied to anti-CD171 - immoblilized ELISA wells, followed by dilution series of anti-DD probes (full strength (f.s.), Finlayson Docket No.: 2200-1405

[0143] 1Z>, and 14 dilution) to show dose dependent anti-DD reaction (Fig. 4D). Such probe reaction was blocked when DD antigen (0.14 mg / mL) was included in the probe solution (Fig. 4D, left).

[0144] Quantification of plasma levels of DD+NDE.

[0145] We first identified high titer plasma concentrations of CD171+DD+(DD+NDE) and assigned quantification standard as 100 units / mL (LJ / mL). Standard plasma demonstrated a linear dilution curve (Figure 5A). Using this standard curve, plasma levels of DD+NDE were quantified. As in prior studies (2-3), concentrations were widely different among subjects, and no significant difference was found between control and patients with ICH (Figure 5B).

[0146] Specifically, Figure 5 shows the quantification of plasma levels of DD+NDE. We first identified high titer plasma of the levels of CD171+DD+(DD+NDE), and assigned it as 100 units / mL (U / mL) as a quantification standard. Figure 5A. Dilution curve of standard plasma. Figure 5B. Results of human samples. We measured DD+NDE in 23 different control plasma and patient samples collected 1 , 3, 5, and 7 days after hemorrhagic stroke. ELISA readings were converted to U / mL using the 4 parameter logistic formula of standard dilution curve.

[0147] Post-ICH changes in DD+NDE.

[0148] DD+NDE levels gradually increased from Day 1 values (D U / mL, 11 patients) and peaked around 5-7 days in patients with hematoma volumes size less than 5 mL (Figure 6A), whereas patients with ICH volumes >5mL showed no or slow increases (Figure 6B). Such increases were seen in patients with favorable outcome (Figure 6C) versus those with unfavorable outcome (Figure 6D).

[0149] Specifically, Figure 6 shows post-hemorrhagic stroke changes in DD+NDE. Although plasma levels of DD+NDE were widely different among subjects (Fig. 2B), the levels were quite stable in each subject and demonstrated unique individual post-stroke Finlayson Docket No.: 2200-1405 patterns. Y axis: The changes in DD+NDE (D U / mL) from Day 1 . A-B: stroke size between <5 and >5 mL. C-D: 6 months mRS score between <=4 and >=5.

[0150] Discussion

[0151] This study introduces a novel approach to monitoring local brain biological activity by analyzing proteins and biomolecules presented on the surface of nearby exosomes. Specifically, we focused DD+NDE as a marker of post-ICH fibrinolysis. Importantly, this assay platform is adaptable: by replacing the anti-D-dimer antibody with others, the method could be extended to assess a wide range of local brain responses.

[0152] After ICH, blood clot formation, subsequent fibrinolysis, oxidative stress, neuroinflammation, brain edema, glial scar formation, Wallerian degeneration to remove damaged axons, revascularization, and repair of injured neurons and glial cells occur. The timing, intensity, and nature of these responses can vary between patients and significantly influence clinical outcomes. Despite their importance, no reliable clinical tools are available to assess them in real time, outside of imaging. Monitoring even one of these responses dynamically could open the door to personalized patient management and targeted interventions. Imaging modalities offer promise but are not as well suited for frequent (i.e., greater than daily) monitoring. On the other hand, modem blood tests can detect proteins at extremely low concentrations, even down to the single-molecule level. However, many functional proteins act locally (i.e., in the brain) and are not consistently released into the bloodstream in detectable quantities.

[0153] NDE was first identified in peripheral blood in 2015, a discovery that marked the beginning of a new era in NDE-based biomarker research. Considerable inter-individual variability exists in plasma levels of NDE, which poses a significant challenge for cross- sectional comparisons between patient and control groups. However, within each individual, NDE levels remain remarkably stable over time and show substantial shifts in response to physiological or pathological events. These findings suggest that NDE assays are well-suited for tracking disease progression and evaluating therapeutic responses. In addition to their stability, we also demonstrated that NDEs are capable of Finlayson Docket No.: 2200-1405 capturing a variety of proteins on their surface, offering a versatile platform for molecular profiling of brain-derived signals in blood. The present study on DD+NDE builds directly upon these prior findings, establishing a novel method for monitoring post-hemorrhagic fibrinolytic activity via a blood-based exosomal assay.

[0154] Studies of plasma D-dimer in ICH have found associations between plasma D- dimer levels and adverse outcomes; along with finding that patients with spontaneous ICH with elevated plasma D-dimer were more likely to have larger hemorrhages, intraventricular extension, and early neurological deterioration, while studies have found a correlation between plasma D-dimer levels and poor outcome in patients with traumatic ICH.

[0155] Our results, though in a small sample size, appear to suggest a contrary outcome in DD*NDE levels in atraumatic ICH: if early elevation of D-dimer reflects early hemostasis, this could, in turn control hemorrhage volume, thereby resulting in improved functional outcome. Two factors could account for these differences: firstly, the selectivity of NDE detection versus general plasma levels of D-dimer, whose elevation could suggest a general inflammatory state, and that whereas studies measured plasma levels on day 1 following ICH, our assay allows serial measurement on days 1 -7 to characterize overall temporal patterns of DD*NDE variation within individuals.

[0156] Example 4: Change of exosome marker CD9 on the surface of oligodendrocyte- derived exosomes

[0157] Plasma samples were collected after appropriate Institutional Review Board approval at Duke University. Plasma samples from deep intracerebral hemorrhage (hemorrhagic stroke in white matter) (n=7, 2 male and 5 female, 57+7.7 years old) (Duke University) were collected 1 , 3, 5, and 7 days after stroke with some exception of blood collection timing. The purpose of this study was to evaluate post-stroke acute phase biomarkers and blood samples were collected during the 1stweek after stroke. We only selected patients with white matter lesions (deep intracerebral bleeding). Some patients Finlayson Docket No.: 2200-1405 showed gradual increase in ODE levels, indicating that ODC may be activated to initiate repair cascade. No one showed substantial decrease of ODE.

[0158] Figure 7 shows plasma levels of oligodendrocyte-derived exosomes by measuring CD9+MOG+double positive signals (CD9 on MOG, Y-axis). Deep intracerebral hemorrhage (hemorrhagic stroke in white matter) (n=7, 2 male and 5 female, 57+7.7 years old). Blood samples were collected 1 , 3, 5, and 7 days after stroke with some exception.

[0159] Thus, specific embodiments and methods of use of biomarkers for the assessment of post-hemorrhagic stroke and their uses in various applications have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the disclosure herein. Moreover, in interpreting the specification, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

Claims

1. Finlayson Docket No.: 2200-1405CLAIMS1 . A method of measuring the effects of an intracerebral hemorrhage, comprising: acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage, utilizing the at least one blood, serum, or plasma sample to isolate at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least one surface biomarker, wherein the at least one surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte- derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof, and quantifying the at least one surface biomarker to monitor post-hemorrhage cellular and molecular cascades in the patient.

2. The method of claim 1 , wherein the extracellular vesicle comprises exosomes, ectosomes, and apoptotic bodies.

3. The method of claim 1 , wherein the at least one surface biomarker comprises at least one blood coagulation biomarker, at least one fibrinolysis biomarker, at least one glial scr formation biomarker, at least one antioxidant defense system biomarker, at least one cell recovery biomarker, at least one neuroinflammation biomarker, at least one cell death biomarker, at least one brain edema biomarker, or a combination thereof.

4. The method of claim 3, wherein the at least one blood coagulation biomarker comprises coagulation factors (Factor l-XIII).Finlayson Docket No.: 2200-14055. The method of claim 3, wherein the at least one fibrinolysis biomarker comprises plasmin, plasminogen activators, breakdown products of fibrin, including D-dimer and other Fibrin Degradation Products (FDPs).

6. The method of claim 3, wherein the at least one glial scr formation biomarker comprises Neurocan (NCN), Brevican (BCAN), and Versican (VCAN), Tenascins, Hyaluronic Acid (HA), Fibronectin (FN1 ), Laminin, Collagens, Osteopontin, Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS), Tissue Plasminogen Activator (tPA), Plasmin, Heparanase, Chondroitinase, Cathepsins, and Tissue Inhibitors of Metalloproteinases (TIMPs).

7. The method of claim 3, wherein the at least one antioxidant defense system biomarker comprises superoxide dismutase (SOD3), Glutathione Peroxidase 3 (GPx3), Peroxiredoxin 4 (Prx4), Catalase, Thioredoxin (Trx), Heme Oxygenase-1 (HO-1 ), Paraoxonase 2 (PON2).

8. The method of claim 3, wherein the at least one cell recovery biomarker comprises Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophins, Glial Cell Line-Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Fibroblast Growth Factor (FGF), Insulin-Like Growth Factor-1 (IGF-1 ), Transforming Growth Factor-B (TGFB), Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGE), Neurturin (NRTN), Neuregulin, and Artemin.

9. The method of claim 3, wherein the at least one neuroinflammation biomarker comprises cytokines and chemokines.

10. The method of claim 3, wherein the at least one cell death biomarker comprises ubiquitin carboxy-terminal hydrolase L1 (UCHL1 ), neurofilaments, synaptic proteins, Microtubule-Associated Proteins (MAPs), Neuronal Nuclei Protein (NeuN), Growth-Associated Protein 43 (GAP-43), Calbindin, Parvalbumin, Neurogranin, 15 Postsynaptic Density Protein 95 (PSD-95), Drebrin, a-Synuclein (SNCA), TAR DNA Binding Protein 43 (TDP-43), Neuron-Specific Enolase (NSE),Finlayson Docket No.: 2200-1405 glial fibrillary acidic protein (GFAP), S100 Calcium Binding Proteins (S100B), Glutamine Synthetase (GS), Vimentin, and Astrocytic Phosphoprotein (AP).11 . The method of claim 3, wherein the at least one brain edema biomarker comprises aquaporin 4 (AQP4), Albumin, Immunoglobulins, Fibrinogen, a1 -Antitrypsin, a2- Macroglobulin, Transferrin, Haptoglobin, Ceruloplasmin, C-reactive Protein (CRP), Complement Proteins, Lipoproteins, Clotting Factors, Beta-globulins, Gammaglobulins, Myoglobin, Hypoxia-inducible factor 1 -alpha (HIF-1a), vascular endothelial growth factor (VEGF).

12. The method of claim 1 , wherein acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage includes acquiring the at least one blood, serum, or plasma sample from the brain of a patient.

13. The method of claim 1 , wherein the at least one astrocyte-derived extracellular vesicle comprises excitatory amino acid transporter 1 or 2, or a combination thereof.

14. The method of claim 1 , wherein the at least one oligodendrocyte-derived extracellular vesicle comprises myelin oligodendrocyte glycoprotein (MOG), or a combination thereof15. The method of claim 1 , wherein the at least one neuron-derived extracellular vesicle comprises CD171 .

16. The method of claim 1 , wherein the method is also a measure of the status of post- intracranial hemorrhage cellular stress, death, and recovery, thrombosis and fibrinolysis, hemorrhage expansion and brain edema, inflammation, or a combination thereof.

17. The method of claim 1 , wherein the intracranial hemorrhage is atraumatic.

18. A method of measuring the effects of brain trauma and degree of recovery, comprising: acquiring at least one blood, serum, or plasma sample from a patient suffering from a brain trauma,Finlayson Docket No.: 2200-1405 neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least one surface biomarker, wherein the at least one surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte- derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof, and quantifying the at least one surface biomarker to monitor a cellular cascade and a molecular cascade in the patient.

19. The method of claim 18, wherein the extracellular vesicle comprises exosomes, ectosomes, and apoptotic bodies.

20. The method of claim 18, wherein the at least one surface biomarker comprises at least one blood coagulation biomarker, at least one fibrinolysis biomarker, at least one glial scr formation biomarker, at least one antioxidant defense system biomarker, at least one cell recovery biomarker, at least one neuroinflammation biomarker, at least one cell death biomarker, at least one brain edema biomarker, or a combination thereof.

21. The method of claim 20, wherein the at least one blood coagulation biomarker comprises coagulation factors (Factor l-XIII).

22. The method of claim 20, wherein the at least one fibrinolysis biomarker comprises plasmin, plasminogen activators, breakdown products of fibrin, including D-dimer and other Fibrin Degradation Products (FDPs).

23. The method of claim 20, wherein the at least one glial scr formation biomarker comprises Neurocan (NCN), Brevican (BCAN), and Versican (VCAN), Tenascins, Hyaluronic Acid (HA), Fibronectin (FN1 ), Laminin, Collagens, Osteopontin, Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS), Tissue Plasminogen Activator (tPA), Plasmin,Finlayson Docket No.: 2200-1405Heparanase, Chondroitinase, Cathepsins, and Tissue Inhibitors of Metalloproteinases (TIMPs).

24. The method of claim 20, wherein the at least one antioxidant defense system biomarker comprises superoxide dismutase (SOD3), Glutathione Peroxidase 3 (GPx3), Peroxiredoxin 4 (Prx4), Catalase, Thioredoxin (Trx), Heme Oxygenase-1 (HO-1 ), Paraoxonase 2 (PON2).

25. The method of claim 20, wherein the at least one cell recovery biomarker comprises Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophins, Glial Cell Line-Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Fibroblast Growth Factor (FGF), Insulin-Like Growth Factor-1 (IGF-1 ), Transforming Growth Factor-B (TGFB), Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGE), Neurturin (NRTN), Neuregulin, and Artemin.

26. The method of claim 20, wherein the at least one neuroinflammation biomarker comprises cytokines and chemokines.

27. The method of claim 20, wherein the at least one cell death biomarker comprises ubiquitin carboxy-terminal hydrolase L1 (UCHL1 ), neurofilaments, synaptic proteins, Microtubule-Associated Proteins (MAPs), Neuronal Nuclei Protein (NeuN), Growth-Associated Protein 43 (GAP-43), Calbindin, Parvalbumin, Neurogranin, 15 Postsynaptic Density Protein 95 (PSD-95), Drebrin, a-Synuclein (SNCA), TAR DNA Binding Protein 43 (TDP-43), Neuron-Specific Enolase (NSE), glial fibrillary acidic protein (GFAP), S100 Calcium Binding Proteins (S OB), Glutamine Synthetase (GS), Vimentin, and Astrocytic Phosphoprotein (AP).

28. The method of claim 20, wherein the at least one brain edema biomarker comprises aquaporin 4 (AQP4), Albumin, Immunoglobulins, Fibrinogen, a1 - Antitrypsin, a2-Macroglobulin, Transferrin, Haptoglobin, Ceruloplasmin, C-reactive Protein (CRP), Complement Proteins, Lipoproteins, Clotting Factors, Betaglobulins, Gamma-globulins, Myoglobin, Hypoxia-inducible factor 1 -alpha (HIF- 1 a), vascular endothelial growth factor (VEGF).Finlayson Docket No.: 2200-140529. The method of claim 18, wherein acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage includes acquiring the at least one blood, serum, or plasma sample from the brain of a patient.

30. The method of claim 18, wherein the at least one astrocyte-derived extracellular vesicle comprises excitatory amino acid transporter 1 or 2, or a combination thereof.

31. The method of claim 18, wherein the at least one neuron-derived extracellular vesicle comprises CD171 .

32. The method of claim 18, wherein the method is also a measure of the status of post-intracranial hemorrhage cellular stress, death, and recovery, thrombosis and fibrinolysis, hemorrhage expansion and brain edema, inflammation, or a combination thereof.

33. The method of claim 18, wherein the intracranial hemorrhage is atraumatic.

34. A method of measuring the effects of an intracerebral hemorrhage, comprising: acquiring at least one blood, serum, or plasma sample from a patient suffering from intracerebral hemorrhage, utilizing the at least one blood, serum, or plasma sample to isolate at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, at least one astrocyte-derived extracellular vesicle, or a combination thereof to form an extracellular vesicle isolate composition, analyzing the exosome isolate composition to identify at least a first surface biomarker and at least a second biomarker, wherein the first surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte- derived extracellular vesicle, or the combination thereof and wherein the second surface biomarker is located on the surface of the at least one neuron-derived extracellular vesicle, at least one oligodendrocyte-derived extracellular vesicle, the at least one astrocyte-derived extracellular vesicle, or the combination thereof andFinlayson Docket No.: 2200-1405 is also a coagulation biomarker, a fibrinolysis biomarker, an inflammation biomarker, or a combination thereof, and quantifying the first surface biomarker and the second biomarker to monitor posthemorrhage cellular and molecular cascades in the patient.

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