WO2014124174A2 - Circulating bmec and related cells as biomarkers of cns diseases associated with the blood-brain-barrier disorders - Google Patents

Abstract

Described herein are processes, assays and methods for detecting damage to the blood-brain barrier by assaying the levels of any one or more of cBMEC, EPC and/or UCHL1. An increase in the levels of cBMEC, EPC and/or UCHL1 in a sample obtained from the subject is indicative of damage to the blood-brain barrier.

Description

CIRCULATING BMEC AND RELATED CELLS AS BIOMARKERS OF CNS DISEASES ASSOCIATED WITH THE BLOOD-BRAIN-BARRIER DISORDERS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/762,794 filed on February 8, 2013, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] The invention was made with government support under Grant Nos. A 1040635, DA034515 and NS047599 awarded by the National Institutes of Health. The government has certain rights to the invention.

TECHNICAL FIELD

[0003] The invention relates to processes, assays and methods for detecting central nervous system (CNS) disorders, such as damage to the blood-brain barrier (BBB) and method for treating the same.

BACKGROUND

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] Central nervous system (CNS) disorders, including traumatic brain injury and blood-brain barrier (BBB) damage caused by microbial infection (e.g., NeuroAIDS), stroke, drug abuse, brain tumor and neurodegenerative diseases, remain the world's leading causes of disabilities despite aggressive research [1]. Because the brain is the most delicate organ of the body that is protected by the BBB, which constitutes the largest surface area [2], the diseases associated with BBB disorders account for more hospitalizations and prolonged care than almost all other diseases combined. The patients experiencing devastating CNS diseases far outnumber those suffering and dying from all types of systemic cancers or heart diseases [1]. Despite significant advances in highly active antiretroviral therapy (HAART), the prevalence of neuroAIDS has significantly increased [3]. This is mainly due to the inability of antiretroviral drugs to cross the BBB [3] and the role of CNS as the reservoir for HIV-1, which is capable of migrating out of the brain [4]. The incidence of NeuroAIDS is higher or accelerated among the aging populations and drug users [5]-[6]. Over one -third of the entire population will experience a CNS disorder during their lifetime [1]. The incidence of CNS disorders also increases with age. All these factors together, along with the fact that there are no surrogate markers available for the BBB injury, exacerbate the problem of diagnosis/prognosis, prevention and treatment of the CNS disorders.

[0006] Quantitative evaluation of the BBB injury has been one of the most challenging issues in the CNS disorders caused by microbial (e.g., meningitic pathogens) and non- microbial (e.g., methamphetamine and nicotine) insults [l]-[3], [6]-[18]. Successful isolation and cultivation of BMECs, which are the relevant in vitro model of the BBB, has enabled us and others to perform both molecular characterization and genomewide analysis of the pathogenic mechanisms of microbial and non-microbial factor-caused BBB disorders in vitro (7-18). However, it is difficult to carry out genomewide, noninvasive evaluation of the in vivo BBB injury. A variety of methods have been used to evaluate the function of the BBB in vivo. Leakage of peripheral proteins (e.g., fibrinogen and albumin) into the CNS has been used to evaluate BBB permeability associated with viral encephalitis and other CNS infection [19]. While these techniques have the advantage of using endogenous proteins, the BBB disruption may not be correlated with the protein levels in CNS due to certain nonspecific effects [19]. Recently, magnetic resonance (MRI)-based molecular imaging technologies have gained increasing attention in neuroscience [20]. Although an increasing number of synthesized molecular imaging agents have been tested in vitro, very few have been validated in the brains of live animals. The major challenges in molecular neuroimaging approaches are the poor ability of delivering agents across the BBB [20]. Additional methods involve the injection of dyes, such as Evans blue and sodium fluorescein (NaFI), into a variety of animal model systems for evaluation of BBB permeability [19]. The major limitation of these techniques is that they cannot be used for humans.

[0007] Recently, qualification of circulating endothelial cells (CECs) in peripheral blood has been developed as a novel and reproducible approach for assessing endothelial damage/dys function caused by cardiovascular disorders and inflammatory diseases [21]-[24]. The first descriptions of methods used to detect circulating cells in the blood with endothelial characteristics were reported in the mid-1970s [24]. These methods included density centrifugation, vital light microscopy and histologic staining, which did not isolate and identify CECs reliably. It was two more decades before reliable procedures were developed to detect this rare cell population. Currently, the most common CEC qualification procedures include an enrichment step through immunomagnetic separation of cells using magnetic beads coupled to an antibody against an endothelial antigen such as CD 146 (endothelial marker) or CD34 (progenitor cell marker) [24]. Among endothelial cells circulating in the blood, some are terminally differentiated mature cells (CECs) while others show progenitorlike phenotype [endothelial progenitor cells (EPCs)], suggesting that EPCs may participate in the generation of new vessels through homing to sites of angiogenesis [24]-[26]. Over the past decade increased CECs have been detected in many pathological conditions, including cancer and heart diseases [21]-[26]. Such cell-based biomarkers, however, are not specifically identified for BBB disorders caused by CNS infection and inflammation. Since the BBB is mainly constituted by the specific endothelial cells, called BMECs, it seems plausible that circulating BMECs (cBMECs) could be biomarkers for BBB dysfunctions. Based on our longstanding interest and studies in the BBB injury and CNS disorders, we have hypothesized that cBMECs, which are endowed with a full-blown BBB phenotype, are dynamically shedding from the brain microvasculature upon pathophysiological changes in the CNS. Circulating BMECs can be monitored by experimental approaches and used as noninvasive blood biomarkers in indexing BBB injury, which is caused by meningitic pathogens and other pathogenic insults. In this report, using animal model systems, we have demonstrated for the first time that BBB injury could be detected by the technologies for characterization and quantification of cBMECs derived from the CNS disorders in mice caused by microbial (gpl20 and E. coli Kl) and non-microbial (methamphetamine and nicotine) insults. Furthermore, we have also demonstrated that alpha7 nAChR, an essential regulator of inflammation [14], plays an important role in cBMEC shedding associated with BBB injury caused by nicotine and meningitic E. coli Kl .

SUMMARY

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. [0009] Provided herein is a process that includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of circulating brain micorvascular endothelial cells (cBMEC) and determining the subject has an increased likelihood of CNS disorder if level of cBMEC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample. In some embodiments, the process may further include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample. In an embodiment, an increase in both cBMEC levels and EPC levels in the subject is indicative of increased likelihood of CNS disorder.

[0010] Also provided herein is a process that includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of endothelial progenitor cells and determining the subject has an increased likelihood of CNS disorder if level of EPC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample. The process may further include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample. In an embodiment, an increase in both cBMEC levels and EPC levels in the subject is indicative of increased likelihood of CNS disorder.

[0011] Also provided herein is a process that includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of ubiquitin C-terminal hydrolase 1 (UCHL1) and/or Slurp- 1 and determining the subject has an increased likelihood of CNS disorder if level of UCHL1 and/or Slurp-1 is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of UCHL1 and/or Slurp-1 is the same as or decreased relative to the reference sample. The process may further include the steps of assaying the sample to determine the levels of circulating brain micorvascular endothelial cells and determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample. The process may also include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0013] Figure 1 depicts in accordance with various embodiments of the invention, triple staining (TS) of murine cBMECs (A-E) isolated by the use of UEA magnetic beads. TS was done by DAPI (blue)/antibodies against CD 146 (FITC/green) (for EC) and S100B (for brain) (rhodamine/red) (A-D: cBMECs, CD146+/S100B+/DAPI+). Cells indicated with arrows are cBMECs (A-E) from mice treated with PBS (A: Control), NT (B), METH (C), gpl20 (D), and meningitic E. coli Kl E44 (E).

[0014] Figure 2 depicts in accordance with various embodiments of the invention, BBB disorders induced by NT, METH, and gpl20. Triple staining (TS) of murine cBMECs and EPCs was done by DAPI (blue)/antibodies against CD 146 (FITC/green) (for EC) and S100B (for brain) (rhodamine/red) (cBMECs, CD146+/S100B+/DAPI+) or CD133 (for PC/rhodamine/red) (EPCs, CD146+/CD133+/DAPI+). cBMECs and EPCs were counted with six random fields. Number of total ECs (CEC) (A), cBMECs (B) and EPCs (C) in peripheral blood (ml). Quantification of albumin in CSF (D). (**P<0.001).

[0015] Figure 3 depicts in accordance with various embodiments of the invention, blood levels of UCHL1 (A), S100B (B), CECs (C), cBMECs (D) and EPCs (E) in mice treated with PBS (CON), nicotine (NT), gpl20 (GP) and nicotine+gpl20 (NT+GP).Bars denote mean values, and error bars describe SEM. **P<0.01 & *** PO.001 compared with the control (PBS). [0016] Figure 4 depicts in accordance with various embodiments of the invention, enhancement of cBMEC shedding //? vitro by nicotine (NT), METH (MT) and gpl20_^4. Shedding of cBMECs from BMEC monolayers in the upper chambers of Transwells after exposure to nicotine (10 μΜ), METH (10 nM) and gpl20 (50 ng/ml) for 36 . B. cBMEC shedding from the WT BMEC monolayers after exposure to different doses of nicotine. C. cBMEC shedding from WT (white column) and KO (black column) BMEC monolayers treated with METH. Bars denote mean values, and error bars describe SEM. /?<0.0 5; **P<0.01.

[0017] Figure 5 depicts in accordance with various embodiments of the invention, effects of genetic blockage of a7 nACfiR on nicotine-increased BBB permeability and E44 transcytosis. Triple staining (TS) of murine cBMECs isolated by the use of magnetic beads coupled with UEA-I, which specifically binds to EC [35]. CEC and cBMECs were isolated from wildtype (WT) and a7 deficient (KO) murine pups treated with nicotine (NT), E44 or NT plus E44. Cells without treatment were used as a control. TS was done by DAPI (blue)/antibodies against CD146 (FITC/green) (A: CEC) and S100B (for brain) (rhodamine/red) (B: cBMECs, CD146+/S100B+/DAPI+) or CD133 [for Progenitor ECs(PEC)/rhodamine/red] (PEC: CD146+/CD133+/DAPI+)(Figure SI). CECs and cBMECs were counted with six random fields. (*P< 05; **P<0.01; ***P<0.001). CNS inflammation and BBB injury were further confirmed by quantification of PMN (C) and albumin (D) in CSF, which have been extensively used for assessing BBB disruption [31].

[0018] Figure 6 depicts in accordance with various embodiments of the invention, role of cBMECs and EPCs in physiology and pathology of the BBB. During BBB vascular turnover BMECs might be replaced by proliferation of adjacent cells or by maturation of circulating endothelial progenitors (EPCs) generated in the bone marrow. Circulating endothelial cells (CECs) and BMECs (cBMECs) with a mature phenotype, derived from systemic and BBB vessel turnover, respectively, are increased in patients with systemic inflammation and BBB disorders. The role and the frequency of marrow-derived circulating EPCs may vary in different types of CNS inflammation and in different phases of BBB disorders. In addition to cBMECs and EPCs, cerebral angiogenesis might be modulated by some other specialized cells such as astrocytes and pericytes.

[0019] Figure 7 depicts in accordance with various embodiments of the invention, triple staining (TS) of murine EPCs (A-E) isolated by the use of UEA magnetic beads. TS was done by DAPI (blue)/antibodies against CD 146 (FITC/green) (for EC) and CD 133 (for PC/rhodamine/red) (EPC, CD146+/CD133+/DAPI+). Cells indicated with arrows are EPCs (A- D) from mice treated with PBS (A: Control), NT (B), METH (C), and gpl20 (D).

[0020] Figure 8 depicts in accordance with various embodiments of the invention, the roles of Slurp- 1 in E. coli Kl (E44)- or IbeA-induced pathogenicity (A-C). A-B: Effects of Slurp- 1 on E. coli invasion of HBMEC with different doses of proteins (Pr)(A) and treated with ML A (a7 antagonist)(B). BSA was used as a control. Recombinant human Slurp- 1 with a His6-tag at the N-terminus was expressed in E. coli and purified as described previously (14). Treatment with siRNA and NT (24 h exposure), invasion and Western blot were carried out as described (14). The results are expressed as relative invasion, taking the value for the control as 100% (*P < 0.05; **P<0.01). HBMEC were subjected to the following different treatments. (C): IbeA (0.5 μg/ml) for 24 h; and E. coli Kl E44 (1 X 106 CFU) for 4 h. Cells without treatment were used as controls (CON). The recombinant IbeA protein was purified and treated with polymyxin-B agarose to remove contaminated LPS as described in our previous publications (70). Protein extracts from the cytoplasms (C) were analyzed by Western blotting with antibodies against Slurp- 1 and actin (loading control for C).

[0021] Figure 9 depicts in accordance with various embodiments of the invention, that Slurp- 1 is essential for pathophysiological functions of a7 nAChR and positively correlated with E. coli Kl(E44)-induced meningitis in neonatal mice. The control (CON) and E44 groups received i.p. injection of 10 μΐ PBS and 2 X 105 CFU E. coli Kl E44 in 10 μΐ PBS, respectively. Eighteen hours after injection, blood, CSF and brain tissue specimens were obtained as described previously (14). Meningitis was confirmed by positive bacterial culture in CSF of the pups receiving E44 (data not shown). Slurp-1 in the specimens was detected by ELISA (A: blood), Western blot (WB) (B: CSF), immunofluorsecence (IF) staining (C: mouse brain cortex) and immunohistochemical (IH) staining (D: hippocampus). Antibodies (Ab) against mouse Slurp-1 (rabbit Ab) (A-D) and a7 nAChR (rabbit Ab)(C) were obtained from Dr. Hidemi Misawa (71) and Genescript (14), respectively. ELISA, WB, IF and IH were carried out as described in Appendix 2. A-C: WT mouse pups. D: KO= a7 (-/-) pups. (n=6-8). **P<0.01.

[0022] Figure 10 depicts in accordance with various embodiments of the invention, the blockage of Slurp-1 (SLP)-mediated effects on E44-stimulated PMN-like HL-60 migration across HBMECs with MLA. HBMECs were treated with different doses (0.1-2 μg) of proteins (human Slurp-1; BSA: Control) (A-B). Transmigration assays were carried out as described above. Differentiation of HL60 into PMN-like leukocytes was induced by 1.3% DMSO. HBMEC and DMSO-HL60 were preincubated with (+) and without (-) inhibitors of a7 nAChR [MLA for 1 h (B)]. Leukocyte transmigration was triggered by E44 (105 CFU). The results are expressed as relative migration, taking the control (without treatment) values as 100%. Similar results were obtained with human PMN. The error bars represent the means ± S.D. of three experiments performed in triplicate. *P<0.05; **P<0.01; ***P<0.001.

DETAILED DESCRIPTION

[0023] All references cited herein, including the references cited therein, are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are fully explained in the literature. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 3^rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^th ed., J. Wiley & Sons (New York, NY 2001), Sambrook, et al, Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. Hoboken NJ, John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005), Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-3, 4-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, (2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3) provide one skilled in the art with a general guide to many of the terms used in the present application.

[0024] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0025] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy. In some embodiments, the disease condition is a CNS disorder. In some embodiments, the disease condition is blood-brain barrier damage.

[0027] "Subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0028] "Treatment" and "treating," as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented. Examples of treatment include, but are not limited to, active surveillance, observation, surgical intervention, chemotherapy, immunotherapy, radiation therapy (such as external beam radiation, stereotactic radiosurgery (gamma knife), and fractionated stereotactic radiotherapy (FSR)), focal therapy, systemic therapy, vaccine therapies, viral therapies, molecular targeted therapies, or a combination thereof.

[0029] "Patient outcome" refers to whether a patient survives or dies as a result of treatment. A more accurate prognosis for patients as provided in this invention increases the chances of patient survival.

[0030] Despite aggressive research, central nervous system (CNS) disorders, including blood-brain barrier (BBB) injury caused by, for example, microbial infection, stroke, abused drugs [e.g., methamphetamine (METH) and nicotine], and other pathogenic insults, remain the world's leading cause of disabilities. We previously found that dysfunction of brain microvascular endothelial cells (BMECs), which are a major component of the BBB, could be caused by nicotine, meningitic pathogens and microbial factors, including HIV-1 virulence factors gp41 and gpl20. One of the most challenging issues in this area is that there are no available cell-based biomarkers in peripheral blood for BBB disorders caused by microbial and non-microbial insults. The results herein demonstrate that cBMECs as well as EPCs may be used as potential cell-based biomarkers for indexing of BBB injury. [0031] Accordingly, the invention is based, at least in part, on these findings. The present invention addresses the need for indicators for detection of CNS disorders (for example, in subject suspected of having a CNS disorder) such as damage to the BBB and for guiding treatment options. Provided herein are processes, assays and methods for detecting central nervous system (CNS) disorders, such as damage to the blood-brain barrier (BBB) and method for treating the same.

[0032] Specifically, the provided herein is a process that includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of circulating brain micorvascular endothelial cells (cBMEC) and determining the subject has an increased likelihood of CNS disorder if level of cBMEC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample. In some embodiments, the process may further include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample. In an embodiment, an increase in both cBMEC levels and EPC levels in the subject is indicative of increased likelihood of CNS disorder.

[0033] Also provided herein is a process that includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of endothelial progenitor cells and determining the subject has an increased likelihood of CNS disorder if level of EPC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample. The process may further include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample. In an embodiment, an increase in both cBMEC levels and EPC levels in the subject is indicative of increased likelihood of CNS disorder. [0034] Also provided herein is a process that includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of ubiquitin C-terminal hydrolase 1 (UCHL1) and/or Slurp- 1 and determining the subject has an increased likelihood of CNS disorder if level of UCHL1 and/or Slurp-1 is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of UCHL1 and/or Slurp-1 is the same as or decreased relative to the reference sample. The process may further include the steps of assaying the sample to determine the levels of circulating brain micorvascular endothelial cells and determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample. The process may also include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

[0035] Further described herein is an assay for selecting therapy for a subject having CNS disorder, and optionally administering the therapy. The assay includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of circulating brain micorvascular endothelial cells (cBMEC); determining the subject has an increased likelihood of CNS disorder if level of cBMEC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample and selecting a therapy to treat the CNS disorder if the subject has an increased likelihood of CNS disorder. The assay may further include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

[0036] Also provided herein is an assay for selecting therapy for a subject having CNS disorder, and optionally administering the therapy. The assay includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of endothelial progenitor cells, determining the subject has an increased likelihood of CNS disorder if level of EPC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample and selecting a therapy to treat the CNS disorder if the subject has an increased likelihood of CNS disorder. The assay may further include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample.

[0037] Further described herein is an for selecting therapy for a subject having CNS disorder, and optionally administering the therapy. The assay includes the steps of obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder, assaying the sample to determine level of UCHL1 and/or Slurp- 1, determining the subject has an increased likelihood of CNS disorder if level of UCHL1 and/or Slurp- 1 is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of UCHL1 and/or Slurp- 1 is the same as or decreased relative to the reference sample and selecting a therapy to treat the CNS disorder if the subject has an increased likelihood of CNS disorder. The assay may further include the steps of assaying the sample to determine the levels of circulating brain micorvascular endothelial cells and determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample. The assay may also include the steps of assaying the sample to determine the levels of endothelial progenitor cells and determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

[0038] In various embodiments, the CNS disorder is any one or more of blood-brain- barrier damage, traumatic brain injury (TBI), CNS infection, epilepsy, stroke, brain tumor, neurodegenerative disorders, or a combination thereof. In an embodiment, the CNS disorder is blood-brain barrier damage. In exemplary embodiments, the blood-brain barrier damage may be due to diseases such as meningitis, brain abscess, epilepsy, multiple sclerosis, neuromyelitis optica, late-stage neurological trypanosomiasis, progressive multifocal leukoencephalopathy, De vivo disease and/or Alzheimer's diseases.

[0039] In various embodiments, the sample is any one or more of tissue, blood, plasma, cerebrospinal fluids (CSF) or a combination thereof. In an embodiment, the sample is peripheral blood.

[0040] The invention also provides a system for determining the presence of cBMEC and/or EPC in a sample wherein the sample is obtained from a subject suspected of having BBB damage. The system includes a sample analyzer configured to produce a signal when a cBMEC and/or EPC cells are present in a sample obtained from a subject suspected of having BBB damage and a computer sub-system programmed to calculate, based on the levels of cBMEC and/or EPC detected whether the signal is greater than or not greater than a reference value.

[0041] In some embodiments, the computer sub-system is programmed to compare the mRNA (for example, mRNA encoding cell surface proteins on the surface of cBMEC and/or EPC) to determine a likelihood of BBB damage based on an algorithm that classifies the patient as likely to have BBB damage if mRNA expression is increased relative to a reference value and as unlikely to have BBB damage if the mRNA is not increased. In some embodiments, the mRNA encodes UCHL1 and/or Slurp- 1 or variants thereof or combinations thereof.

[0042] Provided herein is a system that includes a sample from a subject desiring a diagnosis of BBB damage; a detection module configured for quantifying cBMEC cells in the sample; a storage module configured for storing the quantity of cBMEC cells in the sample (the sample quantity) and a reference value of cBMEC cells; a computation module configured for comparing the sample quantity and the reference value and for providing a result that the sample quantity is higher than, equal to, or lower than the reference value; and an output module configured for displaying that the subject has BBB damage if the sample quantity is higher than the reference value or that the subject does not have BBB damage if the sample quantity is not higher than the reference value. [0043] Also provided herein is a system that includes an isolated sample from a subject desiring a diagnosis of BBB damage; a detection module configured for quantifying EPC cells in the sample; a storage module configured for storing the quantity of EPC cells in the sample (the sample quantity) and a reference value of EPC cells; a computation module configured for comparing the sample quantity and the reference value and for providing a result that the sample quantity is higher than, equal to, or lower than the reference value; and an output module configured for displaying that the subject has BBB damage if the sample quantity is higher than the reference value or that the subject has no BBB damage if the sample quantity is not higher than the reference value.

[0044] In some embodiments of the systems, the subject is human and is suspected to have BBB damage. In some embodiments, the isolated sample is cells obtained through affinity purifying a blood sample with UEA-I-coated beads. In some embodiments, the detection module is a fluorescence microscope. In various embodiments of the system, the isolated sample is a blood sample. In various embodiments, the detection module is a flow cytometer or a fluorescence microscope. In various embodiments, the sample obtained from the subject is stained with at least one of an anti-CD146 antibody and an anti-SlOOB antibody. The cBMEC cells may be identified by CD146+S100B+ phenotype. In some embodiments, the sample is stained with at least one of an anti-CD45 antibody, an anti-CD31 antibody, and an anti-GGT antibody and cBMEC cells are identified by GGT+CD31+CD45- phenotype. In some embodiments, the sample is stained with at least an anti-CD 146 antibody and an anti-CD133 antibody and EPC cells are identified by CD146+CD133+ phenotype. In various embodiments, the reference quantity of cBMEC cells is the mean or media quantity of cBMEC cells in a population of subjects without BBB damage. In some embodiments, the reference quantity of EPC cells is the mean or media quantity of EPC cells in a population of subjects without BBB damage.

Assays for detecting cBMEC, EPC, UCHL1 and/or Slurp-1

[0045] In various embodiments of the processes, assays, systems and methods described herein, determining the levels of cBMEC and/or EPC comprises quantitating the number of cells present in a sample obtained from the subject and/or the reference sample using both manual and automated methods. In some embodiments, assay for quantitating the levels of cBMEC and/or EPC in the sample include but are not limited to any one or more of magnetic bead extraction (MBE), flow cytometry, measuring electrical resistance, staining, image analysis, assay using a hemocytometer, assay using a hemocytometer equipped with Neubauer grids, spectrophotometry, single cell technologies including microfluidics-based cell manipulation or a combination thereof [Zhong JF et. al. (2008), A microfluidic processor for gene expression profiling of single human embryonic stem cells. Lab Chip. 2008, 8:68- 74; Leslie M (2011). News Focus: The power of One. Science, 331 :24-26; Kalisky T, Quake SR (2011), Single cell genomics. Nat Methods. 8:311-4; Fritzsch FS (2012), Single-Cell Analysis in Biotechnology, Systems Biology, and Biocatalysis. Annu Rev Chem Biomol Eng.]. Additional methods for determining the levels of cBMEC and/or EPC in a sample will be apparent to a person of skill in the art. In exemplary embodiments, systems such as TC20™ Automated Cell Counter from BIO-RAD or SCEPTER™ Handheld Automated Cell Counter from EMD Millipore may be used to determine the levels of cBMEC and/or EPC in a sample obtained from a subject.

[0046] In various embodiments of the processes, assays, systems and methods described herein, determining the levels of cBMEC and/or EPC includes assaying the levels of the cell surface markers expressed on the surface of cBMEC and/or EPC cells. Examples of cell surface markers for cBMEC include but are not limited to S100B, CD31 (P-CAM), CD146, vWF/Factor VIII, gamma-glutamyl transpeptidase(GGT) and Dil-Ac-LDL. Examples of cell surface markers for EPC include but are not limited to CD34, CD133, CD146, Flk-1, Tie2, and VE-Cadherin.

[0047] In various embodiments of the processes, assays, systems and methods described herein, determining the level ofUCHLl or a variant thereof comprises measuring the amount of nucleic acid encoding UCHL1 or a variant thereof present in the sample, measuring the amount of UCHL1 protein or a variant thereof present in the sample, or a combination thereof.

[0048] In various embodiments of the processes, assays, systems and methods described herein, determining the levels of the cells surface markers on cBMEC comprises measuring the amount of nucleic acid encoding the cell surface markers on cBMEC present in the sample, measuring the amount of cells surface proteins on cBMEC present in the sample, or a combination thereof.

[0049] In various embodiments of the processes, assays, systems and methods described herein, determining the levels of the cells surface markers on EPC comprises measuring the amount of nucleic acid encoding the cell surface markers on EPC present in the sample, measuring the amount of cells surface protein on EPC present in the sample, or a combination thereof

[0050] In some embodiments of the processes, assays, systems and methods described herein, analyzing the sample includes measuring the levels mR A that encode UCHL1 or a variant thereof, cells surface markers on cBMEC and/or cell surface markers on EPC, present in the sample with a polynucleotide capable of hybridizing with mRNA specific for UCHL1 or a variant thereof and/or cell surface markers on cBMEC and/or EPC, under stringent hybridization conditions.

[0051] Techniques that may be used to assess the amount of nucleic acid present in the sample include but are not limited to in situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). Preferred hybridization-based assays include, but are not limited to, traditional "direct probe" methods such as Southern blots or in situ hybridization (e.g., FISH and FISH plus SKY), and "comparative probe" methods such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide -based CGH. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches. Probes that may be used for nucleic acid analysis are typically labeled, e.g., with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. The preferred size range is from about 200 bases to about 1000 bases. Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), Pinkel, et al. (1998) Nature Genetics 20: 207-211, and/or Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992).

[0052] Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green.

[0053] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.

[0054] A two-tailed student t-test with unequal variation may be used to measure the differences between the patient's expression of UCHL1 or a variant thereof and/or cell surface markers on cBMEC and/or EPC, and a normal blood sample, or the patient's own blood (matched control), or a reference generated by computer algorithm pooling many control samples, as described herein. A significant difference may be achieved where the p value is equal to or less than 0.05.

[0055] Suitable methods for assaying the expression level of UCHL1 or a variant thereof UCHL1 or a variant thereof and/or cell surface markers on cBMEC and/or EPC include but are not limited to using DNA sequencing, comparative genomic hybridization (CGH), array CGH (aCGH), SNP analysis, mRNA expression assay, RT-PCR, real-time PCR, or a combination thereof. In various embodiments, the assay to detect the nucleic acid encoding UCHL1 and/or Slurp- 1 or assays to detect the protein levels of UCHL1 and/or Slurp- 1, include but are not limited to any one or more of Northern blot analysis, Southern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), radio-immuno assay (RIA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis or a combination thereof.

[0056] Antibodies, both polyclonal and monoclonal, can be produced by a skilled artisan either by themselves using well known methods or they can be manufactured by service providers who specialize making antibodies based on known protein sequences. In the present invention, the protein sequences are known and thus production of antibodies against them is a matter of routine. [0057] For example, production of monoclonal antibodies can be performed using the traditional hybridoma method by first immunizing mice with an antigen which may be an isolated protein of choice or fragment thereof (for example,UCHLl or a fragment thereof or a variant thereof or cBMEC and/or EPC cell surface marker or fragments thereof or variants thereof) and making hybridoma cell lines that each produce a specific monoclonal antibody. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen using, e.g., ELISA or Antigen Microarray Assay, or immuno-dot blot techniques. The antibodies that are most specific for the detection of the protein of interest can be selected using routine methods and using the antigen used for immunization and other antigens as controls. The antibody that most specifically detects the desired antigen and protein and no other antigens or proteins are selected for the processes, assays and methods described herein.

[0058] The best clones can then be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut) where they produce an antibody-rich ascites fluid from which the antibodies can be isolated and purified. The antibodies can be purified using techniques that are well known to one of ordinary skill in the art.

[0059] In the methods and assays of the invention, the presence of any UCHL1 or a variant thereof and/or cell surface markers on cBMEC and/or EPC is determined using antibodies specific for the UCHL1 or a variant thereof and/or cell surface markers on cBMEC and/or EPC marker proteins or a fragments or variants thereof and detecting immunospecific binding of each antibody to its respective cognate marker.

[0060] Any suitable immunoassay method may be utilized, including those which are commercially available, to determine the level UCHL1 or a variant thereof and/or cell surface markers on cBMEC and/or EPC according to the invention. Extensive discussion of the known immunoassay techniques is not required here since these are known to those of skill in the art. Typical suitable immunoassay techniques include sandwich enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA), competitive binding assays, homogeneous assays, heterogeneous assays, etc. Various known immunoassay methods are reviewed, e.g., in Methods in Enzymology, 70, pp. 30-70 and 166-198 (1980).

[0061] In the assays of the invention, "sandwich-type" assay formats can be used. Some examples of such sandwich-type assays are described in by U.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. An alternative technique is the "competitive- type" assay. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an analog of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al, U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler, et al.

[0062] The antibodies can be labeled. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, label with a chemiluminescent compound. For example, the detection antibody can be labeled with catalase and the conversion uses a colorimetric substrate composition comprises potassium iodide, hydrogen peroxide and sodium thiosulphate; the enzyme can be alcohol dehydrogenase and the conversion uses a colorimetric substrate composition comprises an alcohol, a pH indicator and a pH buffer, wherein the pH indicator is neutral red and the pH buffer is glycine-sodium hydroxide; the enzyme can also be hypoxanthine oxidase and the conversion uses a colorimetric substrate composition comprises xanthine, a tetrazolium salt and 4,5-dihydroxy-l,3-benzene disulphonic acid. In one embodiment, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, or label with a chemiluminescent compound.

[0063] Direct and indirect labels can be used in immunoassays. A direct label can be defined as an entity, which in its natural state, is visible either to the naked eye or with the aid of an optical filter and/or applied stimulation, e.g., ultraviolet light, to promote fluorescence. Examples of colored labels which can be used include metallic sol particles, gold sol particles, dye sol particles, dyed latex particles or dyes encapsulated in liposomes. Other direct labels include radionuclides and fluorescent or luminescent moieties. Indirect labels such as enzymes can also be used according to the invention. Various enzymes are known for use as labels such as, for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. For a detailed discussion of enzymes in immunoassays see Engvall, Enzyme Immunoassay ELISA and EMIT, Methods of Enzymology, 70, 419-439 (1980). [0064] The antibody can be attached to a surface. Examples of useful surfaces on which the antibody can be attached for the purposes of detecting the desired antigen include nitrocellulose, PVDF, polystyrene, and nylon. The surface or support may also be a porous support (see, e.g., U.S. Patent No. 7,939,342). The assays can be carried out in various assay device formats including those described in U.S. Pat. Nos. 4,906,439; 5,051,237 and 5,147,609 to PB Diagnostic Systems, Inc.

[0065] In some embodiments of the processes, assays, systems and methods described herein, detecting the level of antibodies reactive to UCHLl or a variant thereof and/or cell surface markers on cBMEC and/or EPC includes contacting the sample from the patient with CNS disorder (such as BBB damage) with an antibody or a fragment thereof that specifically binds UCHLl or cell surface markers on cBMEC and/or EPC, forming an antibody-protein complex between the antibody and UCHLl or a variant thereof and/or cell surface markers on cBMEC and/or EPC present in the sample, washing the sample to remove the unbound antibody, adding a detection antibody that is labeled and is reactive to the antibody bound to UCHLl or a variant thereof or cell surface markers on cBMEC and/or EPC in the sample, washing to remove the unbound labeled detection antibody and converting the label to a detectable signal, wherein the detectable signal is indicative of the level of UCHLl or a variant thereof or cell surface markers on cBMEC or EPC in the sample from the patient. In some embodiments, the effector component is a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound. The level of UCHLl or a variant thereof or cell surface markers on cBMEC or EPC may be obtained by measuring a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of UCHLl or cell surface markers in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of CNS disorder (for example, blood-brain barrier damage).

Reference Value

[0066] In various embodiments of the processes, assays, systems and methods described herein, the reference value is based on the levels of cBMEC, EPC and/or ULCH1. In an embodiment, the reference level is in a blood sample. In an embodiment, the reference level is in a peripheral blood sample. In some embodiments, the reference value is the mean or median level of cBMEC in a population of subjects that do not have a CNS disorder. In some embodiments, the reference value is the mean or median level of EPC in a population of subjects that do not have a CNS disorder. In additional embodiments, the reference value is the level of cBMEC in a sample obtained from the subject at a different (for example, an earlier) time point, such as during diagnosis, before treatment, after treatment or a combination thereof. In some embodiments, the reference value is the mean or median level of expression of UCHL1 in a population of subjects that do not have a CNS disorder. In additional embodiments, the reference value is the mean or median level of expression of UCHL1 in a sample obtained from the subject at a different (for example, an earlier) time point, such as during diagnosis, before treatment, after treatment or a combination thereof.

[0067] In various embodiments, the level of cBMEC in a subject (for example, subject with CNS disorder such as BBB damage) compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In various embodiments, the level of cBMEC in a subject (for example, subject with CNS disorder such as BBB damage) compared to the reference value is increased by at least or about 1-fold, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45- fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof.

[0068] In various embodiments, the level of EPC in a subject (for example, subject with CNS disorder such as BBB damage) compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In various embodiments, the level of EPC in a subject (for example, subject with CNS disorder such as BBB damage) compared to the reference value is increased by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100- fold or a combination thereof.

[0069] In various embodiments, the level of UCHL1 in a subject (for example, subject with CNS disorder such as BBB damage) compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In various embodiments, the level of UCHL1 in a subject (for example, subject with CNS disorder such as BBB damage) compared to the reference value is increased by at least or about 1-fold, 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45- fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof.

Therapies

[0070] As described herein, in exemplary embodiments, the blood-brain barrier damage may be due to diseases including but not limited to meningitis, traumatic brain injury, brain abscess, epilepsy, multiple sclerosis, neuromyelitis optica, late-stage neurological trypanosomiasis, progressive multifocal leukoencephalopathy, De vivo disease and/or Alzheimer's diseases. Damage to the blood-brain barrier may also be caused by microbial infections, stroke, abused drugs and/or other pathogenic insults.

[0071] cBMEC and EPC may be used as biomarkers for therapeutic responses. The transient disruption of the BBB has been used as a new approach for efficient delivery of various alkylglycerols into the CNS [Patel MM (2009), Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs. 23:35-58]. The optimal degree of the BBB disruption is essential in monitoring the therapeutic response to those drugs. It can be easily indicated by quantification of cBMEC, which can provide genomewide profiling of BBB 's cellular components, overcoming the limitation of MRI and the inability of molecular imaging agents to cross the BBB [Lelyveld VS (2010), Int J Imaging Syst Technol. 20:71- 79].cBMEC/EPC can be also used as biomarkers for identification/validation of new CNS drug targets and for guiding the optimal dosing of drugs. Examination of cBMEC from and EPC homing to the BBB vasculature, may result in the identification and validation of drug targets specific for the CNS disorders that can cross the BBB without damaging the brain micro vasculature. Defining the optimum dosage and schedule for CNS drugs has proven to be a challenge. It is increasingly recognized that the recommended dose for further exploration of a drug should be the optimal biological drug dose (OBD) rather than the maximum tolerated dose. Changes in cBMEC counts after dose escalation may provide useful insights in establishing the OBD when assessing agents affecting the BBB. EPC may have great potential for use as a cellular therapy for enhancing vascular repair of BBB disorders because EPC are a population of rare cells that circulate in the blood with the ability to differentiate into BMEC. [0072] In some embodiments, if a subject has increased likelihood of BBB damage, the therapeutic dosage may be adjusted, as would be apparent to a person of skill in the art. For example, if BBB damage is due to a bacterial or viral infection, the prescribed therapeutic dosage may be increased or decreased so as to reduce and/or inhibit damage to the BBB.

[0073] In some embodiments, if a subject has increased likelihood of BBB damage, a Src-inhibitor or nonspecific Src family kinase inhibitor (PP2) may be administered immediately optionally after thrombin injections to block brain edema and BBB disruption (for example, see Liu et al. Blood-brain barrier breakdown and repair by Src after thrombin- induced injury, Ann Neurol. 2010 Apr;67(4):526-33; Paul et al. Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke, Nat Med. 2001 Feb;7(2):222-7).

[0074] In some embodiments, if a subject has increased likelihood of BBB damage corticosteroids and glucocorticoids such as cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), aldosterone; glucocorticoid receptor agonist, and glucocorticoid receptor ligand may be administered to the subject (for example, see Fraser, Can a broken barrier be repaired? J Physiol. 2006 June l; 573(Pt 2): 287).

[0075] In some embodiments, if a subject has increased likelihood of BBB damage, Na- K-Cl cotransporter inhibitor such as bumetanide may be administered to the subject (for example, see, O'Donnell et al. Bumetanide inhibition of the blood-brain barrier Na-K-Cl co- transporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke, J Cereb Blood Flow Metab. 2004 Sep;24(9): 1046-56).

[0076] In some embodiments, if a subject has increased likelihood of BBB damage, dietary supplements and restrictions may be recommended, for example, eliminating gluten, alcohol, trans fats and sugars from the diet to help the brain heal, prescribing supplements that can boost vitamin D, glutathione, Vitamin E, Vitamin C and Co-enzyme QlOm DHA fatty acids from Omega-3 oils and/or prescribing anti-inflammatory botanicals for the brain.

[0077] EPC is one of distinct stem cell populations derived from the bone marrow, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), EPCs and very small embryonic-like stem cells (VSELs) (Herzog EL, et. al., Plasticity of marrow- derived stem cells. Blood. 2003;102:3483-93; Munoz-Elias G, et al, Marrow stromal cells, mitosis, and neuronal differentiation: stem cell and precursor function. Stem Cells. 2003;21 :437-48.). EPCs have been shown to have a great therapeutic potential for a variety of cardiovascular diseases including atherosclerosis, diabetic heart disease, pulmonary hypertension rheumatic diseases, (Reynolds JA, et al, Improving cardiovascular outcomes in rheumatic diseases: Therapeutic potential of circulating endothelial progenitor cells. Pharmacol Ther. 2013 Dec 12. pii: S0163-7258(13)00247-7; Sethi R, Lee CH. Endothelial progenitor cell capture stent: safety and effectiveness. J Interv Cardiol. 2012 Oct;25(5):493- 500). EPCs may play an important role in repair of BBB injury caused by microbial and non-microbial factors since they belong to a population of cells with novel properties capable of angiogenesis and vasculogenesis.

[0078] Genetic engineering of EPCs ex vivo may be used as a promising cell- enhancement strategy for restoring the angiogenic potential of autologous, patient-derived cells (Lavoie JR, Stewart DJ. Genetically modified endothelial progenitor cells in the therapy of cardiovascular disease and pulmonary hypertension. Curr Vase Pharmacol. 2012 May;10(3):289-99). In view of EPCs' potential therapeutic application in BBB repair, the choice of cBMEC as cellular indexing biomarkers for CNS disorders may provide important tools for the structural and functional evaluation of BBB repair.

EXAMPLES

[0079] The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention.

[0080] One of the most challenging issues in this area is that there are no available cell- based biomarkers in peripheral blood for BBB disorders caused by microbial and non- microbial insults. To identify such cellular biomarkers for BBB injuries, our studies have shown that mice treated with nicotine, METH and gpl20 resulted in increased blood levels of CD146+(endothelial marker)/S100B+ (brain marker) circulating BMECs (cBMECs) and CD133+[progenitor cell (PC) marker]/CD146+ endothelial PCs (EPCs), along with enhanced Evans blue and albumin extravasation into the brain. Nicotine and gpl20 were able to significantly increase the serum levels of ubiquitin C-terminal hydrolase 1 (UCHL1) (a new BBB marker) as well as S100B in mice, which are correlated with the changes in cBMECs and EPCs. Nicotine- and meningitic E. coli Kl -induced enhancement of cBMEC levels, leukocyte migration across the BBB and albumin extravasation into the brain were significantly reduced in alpha7 nACfiR knockout mice, suggesting that this inflammatory regulator plays an important role in CNS inflammation and BBB disorders caused by microbial and non-microbial factors. These results demonstrated that cBMECs as well as EPCs may be used as potential cell-based biomarkers for indexing of BBB injury.

Example 1

Experimental Methods

Chemicals and Reagent

[0081] Nicotine tartrate (NT) and methamphetamine (METH) were purchased from Sigma- Aldrich (St. Louis, MO). Dynabeads M-450 Tosylactivated was obtained from Invitrogen (Carlsbad, CA). Ulex europaeus I (UEA I) lectin and mounting medium with 4',6- diamidino-2-phenylindole (DAPI) were purchased from Vector (Buringame, CA). Gpl20 was purchased from Immunodiagnostics (Bedford, MA). Serum levels of molecular markers were determined by ELISA using antibodies and antigens from Creative Biomart (New York, NY) (S100B) and ProteinTech (Chicago, IL) (ubiquitin C-terminal hydrolase 1, UCHLl). All primary antibodies (Ab) were purchased from the commercial sources: a rabbit anti-a7 nACfiR Ab from Genescript (Piscataway, NJ); a rat anti-mouse Ly-6G (Gr-1) Ab; a mouse anti-CD44 Ab (sc-7297), a rabbit anti-P-actin (sc-7210), and a rabbit anti-GGT Ab (sc- 20638) from Santa Cruz Biotechnology (Santa Cruz, CA); a rat anti-mouse Ly-6G (Gr-1) Ab FITC-conjugated and an anti-mouse CD 146 Ab FITC-conjugated from eBiosciences (San Diego, CA), a rabbit anti-SlOOB Ab rhodamine-conjugated from BD Biosciences, and a rabbit anti-CD 133 Ab rhodamine-conjugated from Abbiotec (San Diego, CA). Transwell filters (3 μιη pore size, 6.5 mm diameter), blood plates and CBA assay kit were purchased from BD Biosciences (San Jose, CA).

Animal Model and Treatment Protocol

[0082] All animal experiments were performed using C57BL/6J mice after approval from the IACUC of The Saban Research Institute of Children's Hospital Los Angeles. Heterozygous (+/-) a7-deficient mice with the C57BL/6J background (B6.129S7- Chrna7^tmlBay/J) were purchased from Jackson Laboratory (Bar Harbor, ME). Genotypes of a7^+/+ mice (WT mice), a mice (KO mice) and heterozygous a7^+/~mice were determined according to the PCR protocol provided by the vendor. The animals were used in transgenic breeding at 8 weeks of age for optimum reproductive performance. Male heterozygous (+/^_) and female homozygous (^_/^_) were used in breeding. The average litter size for neonatal mice was 6-8. Age- and sex-matched mice were used in all experiments. Three experiments were carried out. In Experiment 1, WT mice (4 week-old) were divided into 4 groups (I: Control treated with PBS; II: NT; III: METH; and IV: gpl20) (n = 5). Two groups (II and III) of animals were exposed to low dose (1.5 mg/kg/day) of NT (oral delivery) for 3 days (twice per day) or gradually increased doses (2, 4, 6, 8, 10, 10, 10,10, 10, 10 mg/kg from dayl to day 10) of METH [intraperitoneal (i.p.) injection] for 10 days as described previously [59]- [60]. The animals in Group IV received daily injections from tail veins (50 ng/mouse) of endotoxin- free recombinant HIV-1 gpl20 for 2 days as described previously [61]— [62]. The doses of drugs are relevant to the clinical settings of smokers [59], METH abuse [60] and HIV/AIDS (gpl20 in serum 12-92 ng/ml) [63]. To determine if UCHL1 could be used as a novel molecular marker for BBB disorders caused by NT and HIV-1 proteins, Experiment 2 was carried out. Mice (WT) were divided into 4 groups (I: Control treated with PBS; II: NT; III: gpl20; IV: NT+gpl20; n = 4). The animal treatment was performed as described in the first experiment. Serum levels of molecular markers were determined by ELISA using antibodies and antigens from Creative Biomart (New York, NY) (S100B) and ProteinTech (Chicago, IL) (UCHL1). In Experiment 3, the role of a7 nAChR in cBMEC shedding was tested in the neonatal mouse model of E. coli Kl (E44) meningitis using WT (a7^+/+) and KO (a7^~ ) mice. Animals (15 to 20-days old were divided into four groups (I : WT infected with E44; II: WT exposed to NT and infected with E44; III: KO infected with E44; and IV: KO exposed to NT and infected with E44) (6-8 mice/per group). The animals (II & IV) were exposed to NT as described in Experiment 1. After NT exposure, all mice received E. coli Kl strain E44 (2>< 10⁵ CFU) by intraperitoneal injection. Eighteen hours after E. coli inoculation, the animals were anaesthetized with ketamine and lidocaine, and blood samples were collected from heart puncture for bacterial culture using sheep blood plates. After perfusion from heart puncture with 20 ml PBS [64], the skull was opened. CSF samples were collected as described previously [32], [65]. For bacteria counting in CSF, 20 μΐ CSF samples were taken and diluted for bacterial culture with blood plates. For PMN counting in CSF, 50 μΐ CSF samples were stained with a FITC-conjugated rat anti-mouse Ly-6G (Gr-1) antibody and counted under fluorescence microscopy. Albumin concentrations in CSF samples were determined using a mouse Albumin ELISA kit from Bethyl laboratories (Montgomery, TX) according to the manufacturer. Isolation and Counting of Mouse cBMECs

[0083] Mouse cBMECs and BMECs were isolated with Ulex europaeus I (UEA I) lectin- coated Dynabeads as described previously [35]. The beads were prepared according to the manufacturer's instructions (Invitrogen) and resuspended in Hanks' balanced salt solution (HBSS, Invitrogen Corp., Carlsbad, CA, USA) plus 5% fetal calf serum (HBSS+5%FCS) to a final concentration of 4xl0⁸ beads/ml. Mouse CECs, cBMECs and EPCs in whole blood were affinity captured at 4°C with UEA-I-coated Dynabeads. To eliminate non-specific cell binding to the beads, the cell suspensions were flushed through the pipette tip during the washing steps and then suspended in PBS. The cells were transferred to glass splices to by cytospin for staining and counting under a fluorescence microscope. Total ECs or CECs (CD146+/DAPI+), cBMECs (CD146+/S100B+/DAPI+) and EPCs

(CD146+/CD133+/DAPI+) were identified based on their S100B [28] (brain marker)⁺/CD146 [21]-[22] (EC marker)⁺/CD133+ (PC marker)(29-30)/DAPI (nuclei)⁺phenotypes Alternatively, flow cytometry was used for detection of cBMECs in peripheral mouse blood using the following anti-mouse antibodies: CD45-Cy5 (a marker for haematopoietic cells), CD31-APC (a marker for endothelial cells) and CD34-FITC (a marker for Hematopoietic stem cell). A rabbit anti-GGT (gamma-glutamyltranspeptidase) antibody and FITC-conjugated anti-rabbit IgG antibody were used to stain GGT, a marker for brain capillaries. Flow cytometry was carried out as described previously [9] using a FACSCalibur flow cytometer (BD Biosciences) and acquired data analyzed with CellQuest flow cytometry analysis software, with analysis gates designed to remove residual platelets and cellular debris. cBMECs derived from the BBB were identified based on their GGT⁺CD31⁺CD45 phenotype. BMECs were prepared from mouse brain tissues as described previously [14], [35]. Briefly, the mouse (10-day-old) cerebral cortex specimens devoid of large blood vessels were used for isolation of crude microvessels, which were further digested with collagenase (0.1 U/ml), dispase (0.8 U/ml) and DNase I (10 U/ml). Microvascular capillaries were isolated by absorption to Ulex-coated beads. The confluent BMEC monolayer displays a cobblestone appearance when grown on collagen-coated surfaces. The cells were positive for CD 146 [22], demonstrating their endothelial origin, and also expressed S100B [28] and GGT [66], indicating their brain origin. The cells also exhibited the typical characteristics for brain endothelial cells expressing tight junctions and a polarized transport of rhodamine 123, a ligand for P-glycoprotein [67]. Transwell Assays of cBMEC Shedding

[0084] To further investigate cBMEC shedding, the double-chamber Transwell-based in vitro BBB model has been used. BMECs were isolated from WT and a7 nACfiR KO mice as described in our recent publication [14]. BMECs were cultured on collagen-coated Transwell polycarbonate tissue-culture inserts with a pore diameter of 12 μιη (Corning Costar) for 5 days [68]. BMECs were polarized and exhibited a trans-endothelial electrical resistance (TEER) of 200-250 Ω cm ², as measured with an Endohm volt/ohm meter in conjunction with an Endohm chamber (World Precision Instruments) as described previously [68]. After exposure of the BMEC monolayer in the upper chamber to low doses of METH (10 nM) [69], NT (10 μΜ) [10], gpl20 (50 ng/ml) [69] and METH (10 nM)+gpl20 (50 ng/ml) for 36 h, the shed cBMECs in the lower chambers were counted under the microscope. Simultaneously, the integrity of the BMEC monolayer was assessed by measurement of the TEER. Three measurements were made at each time-point for each sample.

Statistical Analysis

[0085] For the analysis of the in vitro data, ANOVA and covariates followed by a multiple comparison test such as the Newmann-Keuls test were used to determine the statistical significance between the control and treatment groups. Software GraphPad Prsim 5.0 was used for analysis of data from animal experiments. P<0.05 was considered to be significant.

Database

[0086] The protein access codes in Swissprot database are listed as follows: a7 nAChR, ra muscularus, Q9JHD6; CD31, ra muscularus, Q08481; CD34, ra muscularus, 064 14; CD45 Mus muscularus, P06800; CD146, ra muscularus, Q8R2Y2; S100B, ra muscularus, V50\ 14; GGT, ra muscularus, Q60928; UCHLl, ra muscularus, P09936.

Example 2

Whole Blood Magnetic Affinity Isolation and Immune-identification of cBMECs as Well as Related ECs

[0087] In order to determine whether cBMECs as well as related ECs are present in peripheral blood and can be used as noninvasive blood biomarkers in cellular indexing of BBB injury caused by microbial and non-microbial pathogenic insults, magnetic bead extraction (MBE) was used to quantitatively evaluate BBB injury in mice caused by NT, METH, gpl20 and E44through measuring of cBMECs derived from the brain. Using UEA-I coated beads, ECs in peripheral mouse blood were evaluated as described previously [27]. Total ECs or CECs (CD146+/DAPI+), cBMECs (CD146+/S100B+/DAPI+) and EPCs (CD146+/CD133+/DAPI+) were identified based on their S100B [28] (brain marker)⁺/CD146[21]-[22] (EC marker)⁺/CD133+ (PC marker) [29]-[30]/D API (nuclei)⁺phenotype (Figure 1A-E). Flow cytometry [9] was also used for detection of cBMECs in peripheral mouse blood by using directly conjugated antibodies against CD45- Cy5 (a marker for haematopoietic cells), CD31-APC (a marker for ECs), CD34-FITC (a marker for Hematopoietic stem cell), and GGT-FITC (gamma-glutamyltranspeptidase) (brain capillaries). There was a good agreement between the two methods for cBMEC quantification (data not shown). Our studies concurred with the literature that there were low blood levels of CECs (<400/ml) (Figure 2A) and EPCs (<40/ml) (Figure 2C) in the control group of animals. We have demonstrated for the first time that very small numbers of cBMECs (<35/ml) could be isolated from the total population of CECs and that significant changes in the levels of cBMECs and EPCs in the peripheral bloodstreams were induced in the animals treated with microbial and non-microbial factors (Figure 2).

Example 2

Levels of cBMECs and EPCs are Significantly Increased in Mice Treated with Nicotine, METH and HIV- 1 gpI20

The levels of CECs, cBMECs and EPCs are significantly higher for the mice treated with drugs (METH or nicotine) and gpl20 when compared to the control (Figure 2A-C), suggesting the involvement of systemic inflammatory response (CEC), the BBB injury (cBMECs) and mobilization of EPCs. Nicotine was able to enhance HIV-1 gpl20-induced cBMEC shedding (Figure 3D). Interestingly, increased numbers of EPCs are correlated with EPC cluster formation induced by nicotine, METH and gpl20 (Figure 7). In order to confirm the reliability of the MBE-based method, quantitative evaluation of METH, NT-, and gpl20- caused BBB injury was carried out by quantification of albumin in CSF, which have been extensively used for assessing BBB disruption [31]— [33]. As shown in Figure 2D, METH, nicotine, and gpl20 were able to significantly increase the BBB permeability to albumin. The similar results were obtained with the Evans blue assays, suggesting that changes in the BBB permeability were correlated with cBMEC shedding. These findings demonstrate that quantification of cBMECs and EPCs by MBE is feasible for evaluating the BBB disruption caused by pathogenic insults.

[0088] Example 3

Changes in cBMEC Levels are Correlated with Alterations of Molecular Markers SIOOB and UCHL1

[0089] To further validate the biological relevance of the cell-based biomarkers, the correlation of cBMECs and molecular biomarkers (S100B and UCHL1) was tested in the mouse model as described in the Experimental Methods. UCHL1, which has a more specific tissue distribution than S100B, is found more exclusively in neurons and is associated with traumatic brain injury [34], but it was unclear whether it could play a role in BBB disorders caused by microbial factors (e.g., gpl20) and drugs of abuse. To determine if there was a correlation between cBMECs and S100B/UCHL1, and if UCHL1 could be used as a novel molecular marker for BBB disorders caused by drugs of abuse {e.g., nicotine) and the HIV-1 proteins such as gpl20, the animal treatment and cBMEC/EPC quantification were performed as described in the first experiment. Serum levels of molecular markers were determined by ELISA using antibodies and antigens from Creative Biomart (New York, NY) (S100B) and ProteinTech (Chicago, IL) (UCHL1). Our results showed that nicotine and gpl20 were able to increase blood levels of both molecular (UCHL1 and S100B) and cellular (cBMECs and EPCs) markers (Fig. 3A-E), suggesting that UCHL1 is a potential new biomarker for BBB disorders caused by drugs of abuse and microbial factors. The combination of treatment with nicotine and gpl20 significantly enhanced the levels of biomarkers when compared to the groups treated with nicotine and gpl20 alone, indicating that nicotine was potentiating the toxic effects of gpl20.

Example 4

In vivo-in vitro Correlation of cBMEC Shedding

[0090] The next set of experiments were designed to address the in vivo-in vitro correlation of cBMEC shedding. The approach exploited an in vitro model in which BMECs were treated with microbial (gpl20) and non-microbial (NT and METH) factors likely to be important in the pathogenesis of BBB disorders. Specifically, BMECs were cultured in the presence or absence of gpl20, NT, METH and gpl20 plus NT or METH (MT). Treatment was followed by counting of cells in the lower chambers for assessment of cBMEC shedding. As seen in Figure 4 A, NT, METH and g l20 could significantly induce cBMEC shedding than the treatment with medium alone (CON). Moreover, significantly higher levels of cBMECs were detected in the combination of g l20 with NT or METH. NT could significantly increased cBMEC shedding in a dose-dependent manner (Figure 4B). In order to establish in vitro models for examining the role of a7 nAChR in cBMEC shedding, wildtype (WT) and a7 nAChR knockout (KO) BMECs were isolated and purified from the brains of 10-day-old wildtype ( 7^+/+) and a7-deficient mice (a7 ^) using UEA I lectin-coated beads as described previously [14], [35]. Most interestingly, METH-induced cBMEC shedding was abolished in a7 nAChR-deficient BMECs when compared to the wildtype cells (Figure 4C).

Example 5 a7 Deficient Animals are Defective in BBB Disorders Caused by Microbial (Meningitic E. coli Kl) and Non-microbial (NT) Factors

[0091] As a7 nAChR plays an important role in CNS inflammation induced by microbial and non-microbial factors [10], [14]— [15], we have proposed that turnover and shedding of BMECs could be regulated by a7 nAChR. To test this hypothesis, the correlation of a7 nAChR with cBMEC shedding and CNS inflammation induced by meningitic E. coli Kl (E44) and nicotine was examined in the gene knockout mouse model of a7 nAChR as described in the experimental methods herein. In this study, wildtype ( 7^+/+) and KO (a7^_/~) neonatal (10 day-old) mice were intraperitoneally injected with E44 after treatment with nicotine for 3 days. As shown in Figure 5, nicotine could significantly increase shedding of both CEC (5 A) and cBMECs (5B) but could not induce any changes in KO mice treated with E44 alone or combined with nicotine (P<0.01). The levels of cBMECs were moderately increased in the WT mice treated with E44 alone, while the combination of E44 with nicotine greatly enhanced cBMEC shedding, suggesting that a7 nAChR plays an essential role in the synergistic effects of nicotine on BBB disorders caused by E44. Similarly, the PMN counts (Figure 5C) and albumin extravasation (Figure 5D) in CSF were significantly reduced in KO mice as compared to wildtype animals (P<0.001). Nicotine was only able to enhance PMN transmigration and albumin extravasation in wildtype mice as compared to corresponding controls (P<0.001), suggesting that a7 nAChR also contributes to the correlation of CNS inflammation with cBMEC shedding. Taken together, these data suggested that a7 nAChR could play an essential role in regulation of CNS inflammation and cBMEC shedding induced by microbial and non-microbial factors.

[0092] Slurp- 1 expressed on BMEC or cBMEC is also a marker for BBB damage. Figure 8shows the roles of Slurp- 1 in E. coli Kl (E44)- or IbeA-induced pathogenicity. Figure 9 shows that Slurp- 1 is essential for pathophysiological functions of a7 nAChR and positively correlated with E. coli Kl(E44)-induced meningitis in neonatal mice. Figure 10 shows the blockage of Slurp- 1 (SLP)-mediated effects on E44-stimulated PMN-like HL-60 migration across BMECs with MLA, which is an antagonist of a7 nAChR. It suggests that PMN transmigration across BMEC is dependent on a7 nAChR/Slurp-1 -mediated signaling and NFKB activation.

[0093] We have developed a new model for the discovery of cell-based BBB biomarkers (Figure 6). Our studies have suggested for the first time that cBMECs can be detected and be used as a cellular index of the BBB damage caused by microbial (E. coli Kl and gpl20) and non-microbial (nicotine and METH) pathogenic insults. The inflammatory response may be regulated by l nAChR. It has been a very challenging issue to directly make the quantification of the BBB injury caused by various pathogenic insults [8]. Most biomarker research into CNS disorders has focused on neuronal damage not on BBB injury, because neuronal sensitivity to certain pathogenic insults is region- and disease-specific [36]. Therefore, much previous research on brain injury has focused on biomarkers that measure neuronal damage. There are a number of clinical advantages for shifting the focus on BBB injury. Firstly most CNS diseases are accompanied by increased BBB dysfunction. Secondly BBB injury does occur concomitantly with the pathogenic insult, but neuronal damage may develop slowly or after a delay. The early detection of BBB disorders offers a window opportunity for neuroprotective intervention. Another advantage is that the cell-based biomarkers cBMECs along with the single cell profiling (SCP) approaches will make the diagnosis of CNS disorders much easier and acceptable. Recent advances in biotechnologies for proteomic and genomic analysis at single-cell resolution enable a global novel understanding of complex biological processes [37]— [41]. The SCP approaches will allow the study of multiple genes/proteins or entire genomes/proteomes of cBMECs. Considering these advantages, the current studies are primarily designed to lay foundations for the use of cBMECs as cellular biomarkers of the BBB injury, which contributes to various CNS disorders. In the current report, we demonstrated that cBMECs could be used as cell-based biomarkers for BBB disorders caused by microbial (e.g., gpl20 and meningitic E. co/z^'Kl) and non-microbial (e.g., nicotine and METH) factors.

[0094] Interestingly, increased numbers of EPCs are correlated with EPC cluster formation induced by nicotine, METH and gpl20. EPCs, derived from bone marrow, are capable of homing to damaged endothelium and furthermore contribute to re- endothelialization and neovascularization [42]-[43]. A correlation of increased EPC number and cluster formation in peripheral blood is observed in BBB injury caused by pathogenic insults on the basis of our study, consistent with the published reports on EPCs mobilized by myocardial ischemia and EPC cluster formation in cerebral small vessel disease [44]-[45]. There are two mechanisms by which endothelial repair occurs have been recently identified [46]. The lost and damaged cells can be replaced by local replication of adjacent mature endothelial cells. However, if local replication were the dominant mechanism of endothelial repair, it would rapidly lead to loss of endothelial integrity. More recently, it has become clear that maintenance and repair of the endothelium by circulating EPCs is an alternative mechanism. These circulating cells in the peripheral blood are derived from the bone marrow, and can differentiate into mature cells with endothelial characteristics. Our findings suggest that EPCs may actively participate in the repair of BBB.

[0095] BMECs may have antigenic overlap with non-BMECs. To determine the total cBMEC number but not a subpopulation, it is essential to select markers that are specifically and constantly expressed by all cBMECs. So far, there are no such markers that meet these criteria. Therefore, the assays depend on multiple characteristics to detect cBMECs as well as EPCs. UEA-I (for EC), CD146 (for EC), CD133 (for PC), GGT (for brain) and S-100B (for brain) are used as detection markers. UCHL1 (also known as PGP9.5), which is a component of the ubiquitin proteosome system [47]-[48], was first detected as "brain-specific protein" in 1981 [48]. This protein has a more specific tissue distribution than S100B and is found more exclusively in neurons [34]. Increased expression of UCHL1 is associated with mechanical stress-caused vascular damage [49], oncogenesis [50] and traumatic brain injury [34]. UCHL1 acts as an oncogene and is found to be related to lymph node metastasis in colorectal cancer[50]. It belongs to the family of deubiquitinating enzymes (DUBs), which constitute the ubiquitin-dependent proteolytic system (UPS). DUBs are emerging as important regulators of many pathways contributing to regulation of both oncogenes and tumor suppressors [51]. Cancer can be promoted by both overexpression and loss of function of DUBs. The metabolic dysregulation of DUBs may contribute to oncogenesis and inflammatory diseases. However, it was unclear whether UCHL1 was associated with BBB injury caused by microbial factors and drugs of abuse. Our data showed that elevated serum levels of UCHL1 were significantly correlated with increased cBMECs in the animals treated with g l20 and nicotine, suggesting that this protein could be used as a new molecular biomarker for BBB injury caused by microbial and non-microbial factors. Recent studies suggest that UCHL1 acts a component of the UPS and contributes to injury-caused vascular remodeling through modulation of NF-κΒ activity and related signaling pathways [49], [52]. UCHL1 may also contribute to regulation of BBB integrity. While UCHL1 is more specific than S100B, it is also present in non-BMEC vascular ECs [49], [52]. SCP may offer new approaches for identification of specific BBB biomarkers through genomewide analysis of cBMECs.

[0096] Our previous studies demonstrated that both microbial (e.g., E. coli Kl) and non- microbial (e.g., nicotine) factors could up-regulate a7 nAChR and that CNS inflammation induced by these pathogenic insults could be blocked by a7 antagonist-mediated inhibition and genetic knockout of the a7 gene [10],[14]-[15]. In concurrence with these findings, a7 nAChR could be up-regulated by METH and gpl20, which are involved in the pathogenesis of HIV-associated neurocognitive disorder (HAND) [53]-[56]. In this report, we have established that a7 nAChR plays an important role in regulation of BBB integrity in the mouse model. The pathogenic insult-induced cBMEC shedding, which is correlated with increased BBB permeability, is significantly reduced in the a7-deficient mice. These data suggest that up-regulation of a7 nAChR is detrimental to the BBB integrity and function.

[0097] The precise mechanism responsible for the pathogenic insult-mediated increase in BBB permeability and cBMEC shedding during CNS inflammation is unknown. Although it is well-known that proinflammatory factors promote increased BBB permeability, it is unclear how the production of these factors is regulated during CNS disorders. Our previous studies showed that a7 nAChR could directly or indirectly upregulate proinflammatory factors (IL-Ιβ, IL-6, TNFa, MCP-1, MIP-la, RANTES, CD44 and ICAM-1), significantly enhance PMN transmigration into CSF and has a detrimental effect on the permeability of the BBB in the early stages of meningitic infection [14]. Calcium signaling mediated by a7 nAChR is the major regulatory pathway for the CNS inflammatory response to meningitic E. coli infection and nicotine exposure. Using the a7 KO mouse model, we demonstrated that decreased cBMEC shedding was correlated with CNS inflammatory response {e.g., decreased PMN recruitment and albumin leakage into CSF) when compared to that in the wildtype animals. These findings provide insight into an element of host defense previously unknown to contribute to the BBB integrity and cBMEC shedding, but the implications of the cholinergic a7 nAChR pathway for the pathogenesis and therapeutics of BBB disorders and CNS inflammation remain to be explored. Alpha7 nAChR has been found to be able to mediate SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein)- 1-upregulated NF-κΒ through both ionic events (calcium signaling) and activation of protein kinases [57]. Both UCHL1 and S100B are shown to be involved in regulation of NF- KB [49], [52], [58]. It is likely that a7 nACfiR-mediated NF-κΒ signaling may be involved in regulation of both the molecular (UCHL1 and S100B) and cellular (cBMEC shedding) biomarkers during various CNS disorders.

[0098] In conclusion, the blood levels of cBMECs as well as EPCs positively correlate with BBB injury and host inflammatory response during CNS inflammation induced by microbial and non-microbial factors. These results enlighten the potential of these noninvasive cell-based biomarkers in indexing BBB injury and optimize therapeutic options.

[0099] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[00100] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments. [00101] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[00102] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[00103] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[00104] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

[00105] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[00106] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[00107] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.

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Claims

A process, comprising:

(i) obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder;

(ii) assaying the sample to determine level of circulating brain micorvascular endothelial cells (cBMEC); and

(iii) determining the subject has an increased likelihood of CNS disorder if level of cBMEC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample.

The process of claim 1, further comprising:

(i) assaying the sample to determine the levels of endothelial progenitor cells; and

(ii) determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

The process of claim 2, wherein an increase in cBMEC levels and EPC levels in the subject is indicative of increased likelihood of CNS disorder.

A process, comprising:

(i) obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder;

(ii) assaying the sample to determine level of endothelial progenitor cells; and

(iii) determining the subject has an increased likelihood of CNS disorder if level of EPC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

The process of claim 4, further comprising:

(i) assaying the sample to determine the levels of endothelial progenitor cells; and (ii) determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample.

The process of claim 5, wherein an increase in both cBMEC levels and EPC levels is indicative of increased likelihood of CNS disorder.

A process, comprising:

(i) obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder;

(ii) assaying the sample to determine level of ubiquitin C-terminal hydrolase 1 (UCHL1) and/or Slurp- 1; and

(iii) determining the subject has an increased likelihood of CNS disorder if level of UCHLland/or Slurp-1 is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of UCHLland/or Slurp-1 is the same as or decreased relative to the reference sample.

The process of claim 7, further comprising

(i) assaying the sample to determine the levels of circulating brain micorvascular endothelial cells; and

(ii) determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample.

The process of claim 7, further comprising

(i) assaying the sample to determine the levels of endothelial progenitor cells; and

(ii) determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

10. The process of claims 1, 4 and 7, wherein the CNS disorder is any one or more of blood- brain-barrier damage, CNS infection, traumatic brain injury, epilepsy, stroke, brain tumor, neurodegenerative disorders, or a combination thereof.

11. The process of claims 1 , 4 and 7, wherein the CNS disorder is damage to the blood-brain- barrier.

12. The process of claims 1, 4 or 7, wherein the sample is tissue, blood, plasma or a combination thereof.

13. The process of claim 12, wherein the blood is peripheral blood.

14. The process of claims 1 or 4, wherein the assay is any one or more of magnetic bead extraction (MBE), flow cytometry, measuring electrical resistance, image analysis, assay using a hemocytometer, assay using a hemocytometer equipped with Neubauer grids, spectrophotometry, microfluidics-based cell manipulation or a combination thereof.

15. The process of claim 7, wherein the assay comprises detecting the level of nucleic acid encoding UCHLl or a variant thereof, detecting the level of nucleic acid encoding Slurp- 1 or a variant thereof, detecting the levels of UCHLl protein or a variant thereof, detecting the levels of Slurp- 1 protein or a variant thereof, or a combination thereof.

16. The process of claim 12, wherein detecting the level of nucleic acid encoding UCHLl or a variant thereof and/or detecting the level of Slurp- 1 or a variant thereof comprises determining the amount of mRNA encoding UCHLl and/or Slurp- 1 or a variants thereof.

17. The process of claim 12, wherein detecting the levels of UCHLl protein or a variant thereof or detecting the levels of Slurp- 1 protein or a variant thereof comprises detecting the levels of UCHLl and/or Slurp- 1 or a variants thereof using an antibody, a nucleic acid, a peptide or a combination thereof.

18. The process of claims 1 or 4, wherein the reference value is the mean or median level of cBMEC in a population of subjects that do not have a CNS disorder.

19. The process of claims 1 or 4, wherein the reference value is the mean or median level of EPC in a population of subjects that do not have a CNS disorder.

20. The process of claims 1 or 4, wherein the reference value is the level of cBMEC in the sample obtained from the subject at an earlier point in time.

21. The process of claim 7, wherein the reference value is the mean or median level of expression of UCHL1 and/or Slurp- 1 in a population of subjects that do not have a CNS disorder.

22. The process of claim 7, wherein the reference value is the mean or median level of expression of UCHL1 and/or Slurp-1 in the sample obtained from the subject at an earlier point in time.

23. The process of claims 1, 4 or 7, further comprising prescribing a therapy to the subject if the subject has an increased likelihood of CNS disorder.

24. The process of claim 23, wherein the therapy is any one or more of therapeutic monitoring, optimal dosing of drugs, EPC-based repair of BBB disorders or a combination thereof.

25. An assay for selecting therapy for a subject having CNS disorder, and optionally administering the therapy, the assay comprising:

(i) obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder;

(ii) assaying the sample to determine level of circulating brain micorvascular endothelial cells (cBMEC);

(iii) determining the subject has an increased likelihood of CNS disorder if level of cBMEC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample; and

(iv) selecting a therapy to treat the CNS disorder if the subject has an increased likelihood of CNS disorder.

The assay of claim 25, further comprising:

(i) assaying the sample to determine the levels of endothelial progenitor cells; and (ii) determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample

An assay for selecting therapy for a subject having CNS disorder, and optionally administering the therapy, the assay comprising:

(i) obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder;

(ii) assaying the sample to determine level of endothelial progenitor cells;

(iii) determining the subject has an increased likelihood of CNS disorder if level of EPC in the subject is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample; and

(iv) selecting a therapy to treat the CNS disorder if the subject has an increased likelihood of CNS disorder.

The assay of claim 27, further comprising:

(i) assaying the sample to determine the levels of endothelial progenitor cells; and

(ii) determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample.

An assay for selecting therapy for a subject having CNS disorder, and optionally administering the therapy, the assay comprising:

(i) obtaining a sample from a subject desiring to know the likelihood of central nervous system (CNS) disorder;

(ii) assaying the sample to determine level of ubiquitin C-terminal hydrolase 1 (UCHL1) and/or Slurp- 1;

(iii) determining the subject has an increased likelihood of CNS disorder if level of UCHL1 and/or Slurp- 1 is increased relative to a reference sample, or determining the subject has decreased likelihood of CNS disorder if the level of UCHLl and/or Slurp- 1 is the same as or decreased relative to the reference sample; and (iv) selecting a therapy to treat the CNS disorder if the subject has an increased likelihood of CNS disorder.

30. The assay of claim 29, further comprising:

i) assaying the sample to determine the levels of circulating brain micorvascular endothelial cells; and

(ii) determining that the subject has an increased likelihood of CNS disorder if the level of cBMEC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of cBMEC in the subject is the same as or decreased relative to the reference sample

31. The assay of claim 29, further comprising:

(i) assaying the sample to determine the levels of endothelial progenitor cells; and

(ii) determining that the subject has an increased likelihood of CNS disorder if the level of EPC in the subject is increased relative to a reference sample or determining that the subject has a decreased likelihood of CNS disorder if the level of EPC in the subject is the same as or decreased relative to the reference sample.

32. A system, comprising;

an isolated sample from a subject desiring a diagnosis of BBB damage;

a detection module configured for quantifying cBMEC cells in the sample;

a storage module configured for storing the quantity of cBMEC cells in the sample (the sample quantity) and a reference value of cBMEC cells;

a computation module configured for comparing the sample quantity and the reference value and for providing a result that the sample quantity is higher than, equal to, or lower than the reference quantity; and

an output module configured for displaying that the subject has BBB damage if the sample quantity is higher than the reference value or that the subject does not have BBB damage if the sample quantity is not higher than the reference value.

33. The system of claim 32, wherein the subject is suspected of having BBB damage.

34. The system of claim 32, wherein the subject is a human.

35. The system of claim 32, wherein the isolated sample is cells obtained through affinity purifying a blood sample with UEA-I-coated beads.

36. The system of claim 32, wherein the detection module is a fluorescence microscope.

37. The system of claim 32, wherein the isolated sample is stained with at least an anti- CD 146 antibody and an anti-SlOOB antibody, and wherein cBMEC cells are identified by CD146⁺S100B⁺ phenotype.

38. The system of claim 32, wherein the isolated sample is a blood sample.

39. The system of claim 32, wherein the detection module is a flow cytometer.

40. The system of claim 32, wherein the isolated sample is stained with at least an anti-CD45 antibody, an anti-CD31 antibody, and an anti-GGT antibody, and wherein cBMEC cells are identified by GGT⁺CD31⁺CD45^" phenotype.

41. The system of claim 32, wherein the reference value of cBMEC cells is the mean or media quantity of cBMEC cells in a population of subjects without BBB damage.

42. A system, comprising;

an isolated sample from a subject desiring a diagnosis of BBB damage;

a detection module configured for quantifying EPC cells in the sample;

a storage module configured for storing the quantity of EPC cells in the sample (the sample quantity) and a reference value of EPC cells;

a computation module configured for comparing the sample quantity and the reference value and for providing a result that the sample quantity is higher than, equal to, or lower than the reference value; and

an output module configured for displaying that the subject has BBB damage if the sample quantity is higher than the reference quantity or that the subject has no BBB damage if the sample quantity is not higher than the reference quantity.

43. The system of claim 42, wherein the subject is suspected of having BBB damage.

44. The system of claim 42, wherein the subject is a human.

45. The system of claim 42, wherein the isolated sample is cells obtained through affinity purifying a blood sample with UEA-I-coated beads.

46. The system of claim 42, wherein the detection module is a fluorescence microscope.

47. The system of claim 42, wherein the isolated sample is stained with at least an anti- CD 146 antibody and an anti-CD 133 antibody, and wherein EPC cells are identified by CD146⁺CD133⁺ phenotype.

48. The system of claim 42, wherein the isolated sample is a blood sample.

49. The system of claim 1, wherein the reference value of EPC cells is the mean or media quantity of EPC cells in a population of subjects without BBB damage.