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WO2018228875A1 - Micro-rna biomarkers of blood-brain barrier dysfunction - Google Patents

Micro-rna biomarkers of blood-brain barrier dysfunction Download PDF

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Publication number
WO2018228875A1
WO2018228875A1 PCT/EP2018/064813 EP2018064813W WO2018228875A1 WO 2018228875 A1 WO2018228875 A1 WO 2018228875A1 EP 2018064813 W EP2018064813 W EP 2018064813W WO 2018228875 A1 WO2018228875 A1 WO 2018228875A1
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Prior art keywords
bbb
mirna
brain barrier
subject
blood
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PCT/EP2018/064813
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French (fr)
Inventor
Gene BOWMAN
Loïc DAYON
Frederic Raymond
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Nestec S.A.
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Publication of WO2018228875A1 publication Critical patent/WO2018228875A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to biomarkers and biomarker combinations that may be used to determine whether a subject has or is at risk of developing an impaired blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • the invention also relates to methods for identifying individuals with an impaired blood brain barrier or at risk of developing an impaired blood brain barrier concomitant with disease or other conditions and it also provides methods of treatment or prevention of blood brain barrier impairment in said individuals.
  • the blood-brain barrier is a selective barrier that separates circulating blood from the brain.
  • the BBB is comprised of endothelial cells bound together by tight junction proteins that form the blood facing side of the lumen of the small cerebral blood vessels.
  • astrocytes in particular, projections from those cells termed astrocytic feet and pericytes contribute to the structure and function of the BBB.
  • the endothelial cells of the BBB express multiple substrate-specific transport systems that control the transport of nutrients, energy metabolites, and other essential molecules from the blood into the brain and the transport of metabolic waste products from the brains interstitial fluid into the blood (Aspelund A. et al. 2015, J. Exp.
  • the BBB serves as a key homeostatic site of the nervous system since it connects the central nervous system (CNS) systemic circulation, and major systems in the body such as respiratory, renal, hepatic and immune systems (Zhao,Z et al. 2015, Cell 163, 1064-1078).
  • CNS central nervous system
  • BBB dysfunction is considered to be a vascular contributor to the risk for the development of age-related cognitive decline, cognitive impairment and dementia, including Alzheimer's disease and its progression.
  • Imaging tools such as magnetic resonance imaging (MRI), computed tomography (CT) or positron emission tomography (PET) are currently used to identify potential dysfunction of the BBB (van der Haar et al. 2015, 49, 71 -81 ).
  • MRI magnetic resonance imaging
  • CT computed tomography
  • PET positron emission tomography
  • the clinical measurement of cerebrospinal fluid to plasma albumin index is another means for identifying BBB dysfunction, however, it involves invasive sampling of the cerebrospinal fluid. Therefore, there is a need for better, minimally invasive, simplified methods of identifying dysfunctions of the BBB at an early stage.
  • BBB dysfunction in living subjects, in particular in subjects that do not exhibit symptoms of or have not been diagnosed with a cognitive impairment.
  • Early diagnosis of subjects with an impaired BBB may enable therapeutic intervention, which may prevent or reduce the risk of the subject developing conditions associated with an impaired BBB, for example, cognitive impairments such as vascular cognitive impairment, vascular dementia, mild cognitive impairment (MCI), age- related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
  • cognitive impairments such as vascular cognitive impairment, vascular dementia, mild cognitive impairment (MCI), age- related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
  • microRNAs are small, non-coding RNAs around 17-25 nucleotides in length. They are regulatory RNA molecules that function to regulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes. Deregulation of miRNA expression has been shown to have an impact on health and diseases (Wang et al. 2016, J. Cell Phys. 231 :25-30). miRNAs are differentially expressed in different cell types, tissues and fluids. They are considered to be an excellent class of blood-based biomarkers since their expression is highly stable in plasma and serum and they have the advantage over measurement of cerebrospinal fluid in that there are relatively easy to monitor.
  • CSF cerebrospinal fluid
  • blood serum samples from 1 18 adults aged 55 and older to analyse the cross-sectional relationship between blood-based biomarkers and BBB function.
  • BBB dysfunction was defined a priori as a CSF-to-serum albumin ratio greater than or equal to 9.0.
  • the amount of albumin crossing the healthy BBB is strictly regularized. Therefore, an increased albumin ratio is considered to be a measure of BBB impairment.
  • the inventors carried out Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression analysis to select the biomarkers that best classified subjects with BBB impairment. Subsequently, diagnostic accuracy was assessed by calculating area under the receiver operating characteristic (ROC) curve. The inventors determined biomarkers for identifying BBB impairment.
  • LASSO Least Absolute Shrinkage and Selection Operator
  • miRNA biomarkers have been determined as biomarkers for identifying BBB impairment.
  • a miRNA profile of biomarkers has been identified in Table 1 for identifying BBB impairment.
  • the measurement of a particular miRNA biomarker is compared to a control to identify BBB impairment.
  • miRNA biomarkers such as miR-204-5p, miR-501 -5p, miR- 136-3p, miR-34a-5p, have been determined as biomarkers for identifying BBB impairment.
  • miRNA biomarkers such as miR-204-5p, miR-501 -5p, miR- 136-3p, miR-34a-5p, together with other blood-based biomarkers such as phosphatidylinositol-glycan-specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , peroxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp) have been determined as biomarkers for identifying BBB impairment.
  • PHLD phosphatidylinositol-glycan-specific phospholipase D
  • Sr strontium
  • PRG4 proteoglycan 4
  • cholesterol ester 17:1 a peroxiredoxin-2
  • IL-16 interleukin 16
  • Trp tryptophan
  • the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein the at least one miRNA is selected from the group of miRNAs comprising Table 1 and an increased or decreased level of expression of miRNA in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood- brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein the at least one miRNA is selected from the group of miRNAs consisting of: miR 204- 5p, miR 501 -5p, miR136-3p or miR34a-5p and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood- brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein the at least one miRNA is selected from the group of miRNAs consisting of: miR 204- 5p or miR 501 -5p and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood- brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNA in a reference wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNAs in a reference wherein the at least two miRNAs are selected from the group of miRNAs comprising Table 1 and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNA in a reference wherein the at least two miRNAs are selected from the group of miRNAs consisting of miR204-
  • the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNA in a reference wherein the at least two miRNAs are miR204-5p and miR501 -5p and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with an impaired blood-brain barrier with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) wherein in addition to miRNA biomarkers further biomarkers are measured.
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with an impaired blood- brain barrier or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of miRNA in the biological sample from the subject c) comparing the level of expression of miRNA in the sample to a level of expression of the miRNA in a reference d) further measuring additional biomarkers wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of identifying a subject with an impaired blood brain barrier or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of miRNA in the biological sample from the subject c) comparing the level of expression of miRNA in the sample to a level of expression of the miRNA in a reference d) further measuring additional biomarkers wherein the additional biomarkers selected from the group consisting of: PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, and tryptophan in one or more samples obtained from the subject and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood brain barrier
  • BBB impaired blood-brain barrier
  • BBB blood-brain barrier
  • the invention provides that the reference is a sample from a normal healthy subject without blood brain barrier impairment.
  • the invention provides that the reference is a sample from the same subject at a different time point.
  • the invention provides that the biological sample is selected from the group consisting of a bodily fluid, tissue or cell. In another aspect, the invention provides that the bodily fluid sample is a blood sample.
  • the invention provides that the bodily fluid is a serum sample.
  • the invention provides the identification of a subject with an impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) is associated with vascular cognitive impairment, vascular dementia, age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
  • BBB blood- brain barrier
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • the invention provides the identification of a subject with an impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) is associated with any of the following conditions: traumatic brain injury, hypoxic ischemia, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
  • BBB blood- brain barrier
  • BBB impaired blood-brain barrier
  • the invention provides a method of treating or preventing blood-brain barrier (BBB) impairment comprising the steps: a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) by measuring biomarkers of the invention according to the method of the invention described above b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof.
  • the intervention is a dietary intervention.
  • the invention provides a method of treating or preventing blood-brain barrier (BBB) impairment comprising the steps: a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) according to the method of identifying the level of expression of the biomarkers of the invention; and b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof wherein the dietary intervention comprises increasing vitamin B intake by the subject, preferably by administering a vitamin B supplement.
  • BBB blood-brain barrier
  • the invention provides a method of treating or preventing blood brain barrier (BBB) impairment comprising the steps: a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) according to the method of identifying the level of expression of the biomarkers of the invention; and b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof wherein the dietary intervention comprises increasing omega-3 fatty acid intake by the subject, preferably by administering an omega-3 fatty acid supplement.
  • BBB blood brain barrier
  • the miRNA is mammalian miRNA.
  • the miRNA is human miRNA (hsa-miRNA). In one embodiment, the miRNA is selected from Table 1 .
  • the miRNA is selected from miR-204-5p, miR-501 -5p, miR-136-3p, or miR-34a-5p.
  • the miRNA is miR-204-5p and/or miR-501 -5p.
  • miR-204-5p is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • miR-501 -5p is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • miR-136-3p is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • miR-34A-5p is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • the method further comprises determining the level of another blood- based biomarker in a sample from the subject.
  • the method further comprises determining the level of one or more biomarkers selected from the group consisting of: PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, or tryptophan in one or more samples obtained from the subject.
  • PHLD is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • strontium is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • PRG4 is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • cholesterol ester 17:1 is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood- brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • IL-16 is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • BBB impaired blood-brain barrier
  • BBB impaired blood-brain barrier
  • tryptophan is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • BBB impaired blood-brain barrier
  • the method comprises determining the level of miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, and tryptophan in one or more samples obtained from the subject.
  • the level of the one or more biomarkers is compared with one or more reference values.
  • each biomarker level in each sample and the corresponding reference values are determined using the same analytical method.
  • the reference values may be based on values (e.g., averages) of the one or more biomarkers in populations of subjects who have, for example, previously been identified as having normal or impaired blood-brain barriers.
  • the method comprises determining a value that represents the prediction of blood-brain barrier impairment (BBB).
  • BBB blood-brain barrier impairment
  • S blood-brain barrier impairment score
  • the method comprises determining a blood-brain barrier impairment score (S) using the formula:
  • the level of miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, PHLD, strontium, PRG4, cholesterol ester, PRDX2, IL-16, and tryptophan are determined in a plasma or serum sample from a subject.
  • the levels of the one or more biomarkers are determined in one or more plasma or serum samples.
  • the subject is a human.
  • the subject is an ageing human. In another embodiment, the subject is a human over the age of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old. Preferably, the subject is a human over the age of 55 years old.
  • the subject substantially does not exhibit any symptoms of a cognitive impairment.
  • the subject has not been diagnosed with a cognitive impairment.
  • the method is an in vitro method.
  • the invention provides a kit for determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB), wherein the kit comprises one or more biomarker of the invention as disclosed herein.
  • the invention provides a kit for determining whether a subject is at risk of developing a cognitive impairment, wherein the kit comprises two or more biomarkers of the invention as disclosed herein.
  • the invention provides a method of treating or preventing blood-brain barrier (BBB) impairment comprising the steps:
  • BBB blood-brain barrier
  • the invention provides a method of preventing or reducing the risk of a cognitive impairment comprising the steps:
  • the intervention is a dietary intervention.
  • the dietary intervention comprises increasing vitamin B intake by the subject, preferably by administering a vitamin B supplement.
  • the dietary intervention comprises increasing omega-3 fatty acid intake by the subject, preferably by administering an omega-3 fatty acid supplement.
  • the invention provides a method of selecting a modification in lifestyle of a subject comprising the steps:
  • the invention provides a method of selecting a modification in lifestyle of a subject comprising the steps: (a) determining whether the subject is at risk of developing a cognitive impairment according to the method of the invention; and
  • the method further comprises applying the selected modification in lifestyle to the subject.
  • the modification in lifestyle comprises a dietary intervention as disclosed herein.
  • the invention provides a diet product for use in treating or preventing blood- brain barrier (BBB) impairment, wherein the diet product is administered to a subject determined to have an impaired blood-brain barrier or to be at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention.
  • BBB blood- brain barrier
  • the invention provides a diet product for use in preventing or reducing the risk of a cognitive impairment, wherein the diet product is administered to a subject determined to be at risk of developing a cognitive impairment according to the method of the invention.
  • the invention provides the use of a diet product for the manufacture of a medicament for treating or preventing blood-brain barrier (BBB) impairment, wherein the diet product is administered to a subject determined to have an impaired blood-brain barrier (BBB) or to be at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention.
  • BBB blood-brain barrier
  • the invention provides the use of a diet product for the manufacture of a medicament for preventing or reducing the risk of a cognitive impairment, wherein the diet product is administered to a subject determined to be at risk of developing a cognitive impairment according to the method of the invention.
  • the invention provides the use of a diet product for treating or preventing blood-brain barrier (BBB) impairment, wherein the diet product is administered to a subject determined to have an impaired blood-brain barrier (BBB) or to be at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention.
  • BBB blood-brain barrier
  • BBB blood-brain barrier
  • the invention provides the use of a diet product for preventing or reducing the risk of a cognitive impairment, wherein the diet product is administered to a subject determined to be at risk of developing a cognitive impairment according to the method of the invention.
  • the diet product is a vitamin B supplement. In another embodiment, the diet product is an omega-3 fatty acid supplement. In another embodiment, the diet product is a folic acid supplement.
  • the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood- brain barrier (BBB) according to the method disclosed herein.
  • BBB blood-brain barrier
  • BBB impaired blood- brain barrier
  • the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject is at risk of developing a cognitive impairment according to the method disclosed herein.
  • the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood- brain barrier (BBB) given the levels of one or more biomarkers from the user, wherein the biomarkers are selected from the one or more biomarkers as disclosed herein.
  • BBB blood-brain barrier
  • BBB impaired blood- brain barrier
  • the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject is at risk of developing a cognitive impairment given the levels of one or more biomarkers from the user, wherein the biomarkers are selected from the one or more biomarkers as disclosed herein.
  • Figure 1 shows the box plot of the biomarker miR-204-5p comparing normal versus impaired blood-brain barrier function where there is a decrease in miR-204-5p in subjects with impaired blood-brain barrier function.
  • Figure 2 shows the box plot of the biomarker miR-501 -5p comparing normal versus impaired blood-brain barrier function where there is a decrease in miR-501 -5p in subjects with impaired blood-brain barrier function.
  • Figure 3 shows the box plot of the biomarker miR-136-3p comparing normal versus impaired blood-brain barrier function where there is a decrease in miR-136-3p in subjects with impaired blood-brain barrier function.
  • Figure 4 shows the box plot of the biomarker miR-34a-5p comparing normal versus impaired blood-brain barrier function where there is an increase in miR-34a-5p in subjects with impaired blood-brain barrier function.
  • Figure 5 shows the box plot of the biomarker PHLD comparing normal versus impaired blood- brain barrier function where there is an increase in PHLD in subjects with impaired blood-brain barrier function.
  • Figure 6 shows the box plot of the biomarker strontium comparing normal versus impaired blood-brain barrier function where there is a decrease in strontium in subjects with impaired blood-brain barrier function.
  • Figure 7 shows the box plot of the biomarker PRG4 comparing normal versus impaired blood- brain barrier function where there is an increase in PRG4in subjects with impaired blood-brain barrier function.
  • Figure 8 shows the box plot of the biomarker cholesterol ester 17:1 comparing normal versus impaired blood-brain barrier function where there is a decrease in cholesterol ester 17:1 in subjects with impaired blood-brain barrier function.
  • Figure 9 shows the box plot of the biomarker PRDX2 comparing normal versus impaired blood-brain barrier function where there is an increase in PRDX2 in subjects with impaired blood-brain barrier function.
  • Figure 10 shows the box plot of the biomarker IL-16 comparing normal versus impaired blood- brain barrier function where there is an increase in IL-16 in subjects with impaired blood-brain barrier function.
  • Figure 1 1 shows the box plot of the biomarker tryptophan comparing normal versus impaired blood-brain barrier function where there is a decrease in tryptophan in subjects with impaired blood-brain barrier function.
  • Receiver operating characteristic (ROC) curves for diagnosis of blood-brain barrier (BBB) impairment for Reference labelled "REF ref”
  • Best models labelled "meta”
  • AUC area under the curve
  • AUC area under the curve
  • the variables selected in the Best model are: the 4 miRNAs miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, and the other blood-based biomarkers PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, and tryptophan.
  • BBB Blood-brain barrier
  • the blood-brain barrier is a selective barrier that separates circulating blood from the brain.
  • the BBB is comprised of a monolayer of endothelial cells bonded by tight junction proteins that form the small cerebral blood vessel lumen.
  • astrocytes in particular, projections from those cells termed astrocytic feet
  • pericytes contribute to the structure and function of the BBB.
  • the BBB governs entry of all peripherally circulating factors such as water diffusion, some gases and lipid-soluble molecules, and selective transport of other substances, such as glucose, amino acids, and micronutrients that are crucial to neuronal function. Conversely, the BBB protects the brain from the passage of toxic substances that may place the central nervous system (CNS) at risk.
  • the term “impaired blood-brain barrier (BBB)” refers to a BBB that is not functioning correctly as a selective barrier between circulation and the brain. As used herein, the term “impaired blood-brain barrier (BBB)" may be equated with “dysfunctional blood-brain barrier (BBB)".
  • An impaired BBB may occur, for example, in a subject having a higher than normal CSF-to- serum albumin ratio, for example a CSF-to-serum albumin ratio greater than or equal to 5, 6, 7, 8, or 9, preferably greater than or equal to 9.
  • Cognitive impairment for example, in a subject having a higher than normal CSF-to- serum albumin ratio, for example a CSF-to-serum albumin ratio greater than or equal to 5, 6, 7, 8, or 9, preferably greater than or equal to 9.
  • vascular cognitive impairment such as vascular cognitive impairment, vascular dementia, mild cognitive impairment (MCI), age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
  • MCI mild cognitive impairment
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • CSF cerebrospinal fluid
  • BBB dysfunction may also present together with conditions of traumatic brain injury, hypoxic ischemia, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
  • BBB dysfunction therefore appears to be a significant risk factor for the development of cognitive impairments and their progression.
  • cognition refers to the set of mental thinking abilities and domains of attention and processing speed, short and long term memory, working memory, executive functions of planning and flexibility, decision making, judgment and evaluation, reasoning and “computation”, problem solving, comprehension and language.
  • Cognitive impairment refers to a deterioration in one or more these domains of cognition.
  • Levels of and improvements in cognition can be readily assessed by the skilled person using any of a number of validated neuropsychological tests standardized to assess, for example, speed of information processing, executive function and memory.
  • Suitable example tests include Mini Mental State Examination (MMSE), Clinical Dementia Rating (CDR), Cambridge Neuropsychological Test Automated Battery (CANTAB), Alzheimer's Disease Assessment Scale-cognitive test (ADAScog), Wisconsin Card Sorting Test, Verbal and Figural Fluency Test and Trail Making Test.
  • MMSE Mini Mental State Examination
  • CDR Clinical Dementia Rating
  • CANTAB Cambridge Neuropsychological Test Automated Battery
  • ADAScog Alzheimer's Disease Assessment Scale-cognitive test
  • Wisconsin Card Sorting Test Verbal and Figural Fluency Test and Trail Making Test.
  • medical imaging of the brain provides an assessment of brain function.
  • medical imaging techniques used for assessment of brain function include electroencephalography (EEG), magnetoencephalography (MEG), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Magnetic Resonance Imaging (MRI), functional Magnetic Resonance Imaging (fMRI), computerised tomography and long-term potentiation.
  • EEG electroencephalography
  • MEG magnetoencephalography
  • PET Positron Emission Tomography
  • SPECT Single Photon Emission Computed Tomography
  • MRI Magnetic Resonance Imaging
  • fMRI functional Magnetic Resonance Imaging
  • computerised tomography computerised tomography and long-term potentiation.
  • BBB blood brain barrier
  • EEG a measure of electrical activity of the brain, is accomplished by placing electrodes on the scalp at various landmarks and recording greatly amplified brain signals. MEG is similar to EEG in that it measures the magnetic fields that are linked to electrical fields. MEG is used to measure spontaneous brain activity, including synchronous waves in the nervous system.
  • PET provides a measure of oxygen utilisation and glucose metabolism.
  • a radioactive positron-emitting tracer is administered, and tracer uptake by the brain is correlated with brain activity.
  • These tracers emit gamma rays which are detected by sensors surrounding the head, resulting in a 3D map of brain activation.
  • the detected radioactivity occurs as a function of regional cerebral blood flow.
  • an increase in cerebral blood flow and neuronal glucose metabolism can be detected within seconds.
  • Suitable analysis can also be based on neuropsychological testing, general and neurological examinations and individual complaints of cognitive decline (e.g., subjective memory loss).
  • Cognitive impairment may be, for example, interpreted as a statistically significant difference in performance at any time point in a suitable test.
  • vascular dementia results from reduced blood flow to the brain, which damages brain cells.
  • the reduced blood flow can occur for a number of reasons, including narrowing of the blood vessels in the brain (subcortical vascular dementia), stroke (single-infarct dementia) and numerous small strokes (multi-infarct dementia).
  • the reduced blood flow may additionally be caused by Alzheimer's disease, a combination referred to as mixed dementia.
  • vascular dementia Early symptoms of vascular dementia include slowness of thought, difficulty with planning, difficulty with language, problems with attention and concentration, and behavioural changes. The symptoms typically worsen in steps, with intervening stable periods of months or years. Age-related cognitive decline
  • Age-related cognitive decline is the normal, non-pathological reduction in cognitive function that is associated with ageing. Although certain mental functions exhibit little age-related decline (e.g., language, reading and vocabulary skills, some numerical abilities and general knowledge) others decline from middle age (e.g., episodic memory, executive functions, speed of processing and reasoning). The extent to which subjects are affected by age-related cognitive decline varies between individuals.
  • MCI Mild cognitive impairment
  • probable Alzheimer's disease diagnosis requires impairment in at least two cognitive domains and impairment of activities of daily living.
  • AD Alzheimer's disease
  • Alzheimer's disease is caused by atrophy of areas of the brain. Although it is not known what initiates the atrophy, studies have found amyloid plaques, neurofibrillary tangles and acetylcholine imbalances in the brains of Alzheimer's patients. Vascular damage in the brain, which may damage healthy neurons, is also common in Alzheimer's patients.
  • Alzheimer's disease is a progressive condition that affects multiple brain functions. Early signs of the disease usually include minor memory problems, for example forgetting recent events or the names of places and objects. As the disease progresses, memory problems become more severe and additional symptoms can develop, such as confusion, disorientation, difficulty making decisions, problems with speech and language, and personality changes. Parkinson's disease (PD)
  • Parkinson's disease is a condition in which nerve cells in the substantia nigra become progressively damaged. Nerve cells in this area of the brain produce dopamine, which acts as a messenger between the parts of the brain and nervous system that control body movement. Damage to these nerve cells results in a reduction in the amount of dopamine produced in the brain, which has the effect of reducing function in the part of the brain controlling movement.
  • Parkinson's disease Symptoms of the Parkinson's disease include tremors, slow movement, and stiff and inflexible muscles. Parkinson's disease patients may also experience additional symptoms, including depression, constipation, insomnia, anosmia and memory problems. Other Conditions associated with BBB Impairment
  • Traumatic brain injury may be another condition associated with BBB impairment.
  • TBI is a non-congenital insult to the brain from an external mechanical force, possibly leading to permanent or temporary impairment of cognitive, physical, and psychosocial functions, with an associated diminished or altered state of consciousness.
  • impairment of the blood-brain barrier may be associated with any of the following conditions: hypoxic ischemia, transient ischemic attack, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
  • Biomarkers microRNAs are examples of the following conditions: hypoxic ischemia, transient ischemic attack, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
  • miR refers to the mature form of the miRNA
  • MIR refers to the gene that encodes them.
  • miRNA sequences have been deposited in miRBase database (http://www.mirbase.org/).
  • the miRBase database is a searchable database of published miRNA sequences and annotation.
  • Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation.
  • miRBase is managed by the Griffiths-Jones lab at the Faculty of Life Sciences, University of Manchester with funding from the BBSRC. miRBase was previously hosted and supported by the Wellcome Trust Sanger Institute.
  • MicroRNAs are isolated from a body fluid.
  • the miRNA is isolated from blood, plasma, serum, cerebrospinal fluid, urine, saliva, tears, amniotic fluid, colostrum, breast milk, bronchial lavage, peritoneal fluid, pleural fluid, or seminal fluid. In one embodiment, the miRNA is isolated from blood, preferably plasma.
  • the miRNA is human.
  • Table 1 represents the miRNA measured in blood serum:
  • miRNA in miRBase there is the sequence of precursor: pre-miRNA (mir-xxx) and the two sequence of miRNA Mature: 5p and 3p (miR-xxx-3p and miR-xxx-5p) and the prefix for the species of origin: hsa-xxx for Homo sapiens.
  • the human pre-miRNA has the following sequence: hsa-mir-204 MI0000284
  • the human mature 5p miRNA has the following sequence: hsa-miR-204-5p MIMAT0000265 uucccuuuGucAuccuAUGCCu (SEQ. ID No . l ) as listed in Table 1 .
  • the human mature 3p miRNA has the following sequence: hsa-miR-204-3p MIMAT0022693
  • the human pre-miRNA has the following sequence: hsa-mir-501 MI0003185
  • the human mature 5p miRNA has the following sequence: hsa-miR-501 -5p MIMAT0002872 AAuccuuuGucccuGGGUGAGA (SEQ ID No .2 ) as listed in Table 1
  • the human mature 3p miRNA has the following sequence: hsa-miR-501 -3p MIMAT0004774 AAUGCACCCGGGCAAGGAUUCU (SEQ ID No.33) mir-136
  • the human pre-miRNA has the following sequence: hsa-mir-136 MI0000475
  • the human mature 5p miRNA has the following sequence:hsa-miR-136-5p MIMAT0000448
  • the human mature 3p miRNA has the following sequence:hsa-miR-136-3p MIMAT0004606
  • the human pre-miRNA has the following sequence: hsa-mir-34a MI0000268
  • the human mature 5p miRNA has the following sequence: hsa-miR-34a-5p MIMAT0000255 uGGCAGUGucuuAGCUGGuuGu (SEQ ID No.4) as listed in Table 1.
  • the human mature 3p miRNA has the following sequence: hsa-miR-34a-3p MIMAT0004557
  • the miRNA-204-5p is human.
  • the miRNA-204-5p is measured in a blood sample, preferably a plasma sample.
  • the miRNA-204-5p is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • RNA sequence of miRNA-204-5p is the hsa-miR-204-5p MIMAT0000265 UUCCCUUUGUCAUCCUAUGCCU (SEQ. ID o.l) . miRNA-501 -5p
  • the miRNA-501 -5p is human. In one embodiment, the miRNA-501 -5p is measured in a blood sample, preferably a plasma sample.
  • the miRNA-501 -5p is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • RNA sequence of miRNA-501 -5p is hsa-miR-501 -5p MIMAT0002872 AAUCCUUUGUCCCUGGGUGAGA (SEQ ID No .2 ) . miRNA-136-3p
  • the miRNA-136-3p is human.
  • the miRNA-136-3p is measured in a blood sample, preferably a plasma sample.
  • the miRNA-136-3p is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • An example RNA sequence of miRNA-136-3p is the hsa-miR-136-3p MIMAT0004606
  • the miRNA-34-5p is human. In one embodiment, the miRNA-34-5p is measured in a blood sample, preferably a plasma sample.
  • the miRNA-34-5p is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • An example RNA sequence of miRNA-34-5p is hsa-miR-34a-5p MIMAT0000255
  • Phosphatidylinositol-glycan-specific phospholipase D Phosphatidylinositol-glycan-specific phospholipase D
  • Phosphatidylinositol-glycan-specific phospholipase D hydrolyzes the inositol phosphate linkage in proteins anchored by phosphatidylinositol glycans (GPI-anchor) thus releasing these proteins from the membrane.
  • the PHLD is human PHLD.
  • the PHLD is measured in a human blood sample, preferable a plasma sample.
  • PHLD An example amino acid sequence of PHLD is the sequence deposited under NCBI Accession No. NP_001494.2.
  • a further example of the amino acid sequence of PHLD is:
  • PHLD may be processed into a mature form, for example by cleavage of a signal peptide.
  • a further example amino acid sequence of PHLD is:
  • a further examples of amino acid sequence of PHLD isoform 2 is: SEQ ID No.40
  • the PHLD level is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • Strontium is an essential element with the largest proportion of strontium located in the bones (>99%). In biological fluids such as serum or plasma it can be bound to proteins.
  • Strontium is measured in a human blood sample, preferably a serum sample. In one embodiment, the Strontium level is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • Proteoglycan 4 plays a role in boundary lubrication within articulating joints. It prevents protein deposition onto cartilage from synovial fluid by controlling adhesion-dependent synovial growth and inhibiting the adhesion of synovial cells to the cartilage surface.
  • Isoform F plays a role as a growth factor acting on the primitive cells of both hematopoietic and endothelial cell lineages.
  • the Proteoglycan 4 is human PRG4.
  • the PRG4 is measured in a blood sample, preferably a plasma sample.
  • a number of isoforms are known: Isoform A, Isoform B, Isoform C, Isoform D, Isoform E, and Isoform F.
  • PRG4 An example amino acid sequence of PRG4 is the sequence deposited under NCBI Accession No. NP_005798.3.
  • amino acid sequence of PRG4 is: SEQ ID No.41
  • PRG4 may be processed into a mature form, for example by cleavage of a signal peptide.
  • a further example amino acid sequence of PRG4 is:
  • a further example of amino acid sequence of PRG4 isoform B is:
  • amino acid sequence of PRG4 isoform C is:
  • amino acid sequence of PRG4 isoform E is:
  • amino acid sequence of PRG4 isoform F is:
  • Proteoglycan 4 level is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • Cholesterol ester 17:1 is also known as 17:1 cholesteryl ester or CE(17:1 ). It is a cholesterol fatty acid ester or simply a cholesterol ester (CE). Cholesterol esters are cholesterol molecules with long-chain fatty acids linked to the hydroxyl group. They are much less polar than free cholesterol and appear to be the preferred form for transport in plasma and for storage. Cholesterol esters are major constituents of the adrenal glands and they also accumulate in the fatty lesions of atherosclerotic plaques. Cholesterol esters are also major constituents of the lipoprotein particles carried in blood (HDL, LDL, VLDL).
  • the cholesterol esters in high- density lipoproteins are synthesized largely by transfer of fatty acids to cholesterol from position sn-2 (or C-2) of phosphatidylcholine catalyzed by the enzyme lecithin cholesterol acyl transferase (LCAT).
  • LCAT lecithin cholesterol acyl transferase
  • the enzyme also promotes the transfer of cholesterol from cells to HDL.
  • cholesterol esters accumulate in the lipoprotein core, cholesterol is removed from its surface thus promoting the flow of cholesterol from cell membranes into HDL. This in turn leads to morphological changes in HDL, which grow and become spherical. Subsequently, cholesterol esters are transferred to the other lipoprotein fractions LDL and VLDL, a reaction catalyzed by cholesteryl ester transfer protein.
  • 17:1 cholesterol ester is PubChem CID: 24779603 with molecular formula C44H 76 0 2 and InChl Key RLMIGWIAENJHMP-RJRTUNKTSA-N.
  • cholesterol ester 17:1 is measured in a blood sample, preferably plasma sample.
  • cholesterol ester 17:1 is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • Peroxiredoxin-2 P32119 (PRDX2_HUMAN)
  • Peroxiredoxin-2 PRDX2
  • PRDX2 Peroxiredoxin-2
  • PRDX2 is involved in redox regulation of the cell. It reduces peroxides with reducing equivalents provided through the thioredoxin system. It is not able to receive electrons from glutaredoxin and it plays an important role in eliminating peroxides generated during metabolism. It participates in the signalling cascades of growth factors and tumor necrosis factor-alpha by regulating the intracellular concentrations of peroxide.
  • the Peroxiredoxin-2 (PRDX2) is human.
  • the PRDX2 is measured in a blood sample, preferably a plasma sample.
  • PRDX2 An example amino acid sequence of PRDX2 is the sequence deposited under NCBI Accession No. NP_005800.3 A further example of the amino acid sequence of PRDX2 is:
  • PRDX2 may be processed into a mature form, for example by cleavage of a signal peptide.
  • a further example amino acid sequence of PRDX2 is:
  • amino acid sequence of PRDX2 isoform 2 is: SEQ ID No.50
  • PRDX2 level is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • Interleukin 16 IL-16
  • IL-16 Interleukin 16
  • IL-16 lnterleukin-16
  • Isoform 1 may act as a scaffolding protein that anchors ion channels in the membrane.
  • Isoform 3 is involved in cell cycle progression in T-cells. Appears to be involved in transcriptional regulation of SKP2 and is probably part of a transcriptional repression complex on the core promoter of the SKP2 gene.
  • the IL-16 is human IL-16.
  • the IL16 is measured in a blood sample, preferably a serum sample.
  • IL-16 An example amino acid sequence of IL-16 is the sequence deposited under NCBI Accession No. NP_757366.2.
  • a further example amino acid sequence of IL-16 is: SEQ ID No.51
  • IL-16 may be processed into a mature form, for example by cleavage of a signal peptide.
  • a further example amino acid sequence of IL-16 is:
  • a further example of amino acid sequence of IL-16 isoform 2 is: SEQ ID No.53
  • a further example of amino acid sequence of IL-16 isoform 3 is:
  • Tryptophan an essential amino acid in humans and animals, meaning the body cannot synthesize it and thus it must be obtained from the diet. Tryptophan is also a precursor to the neurotransmitters serotonin and melatonin.
  • tryptophan is measured in a human blood sample, preferably plasma sample.
  • tryptophan level is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
  • the level of the individual biomarker species in the sample may be measured or determined by any suitable method known in the art.
  • mass spectrometry MS
  • antibody- based detection methods e.g., enzyme-linked immunosorbent assay, ELISA
  • non-antibody protein scaffold-based methods e.g., fibronectin scaffolds
  • radioimmunoassays RIA
  • aptamer-based methods may be used.
  • Other spectroscopic methods, chromatographic methods, labelling techniques or quantitative chemical methods may also be used.
  • the level of the one or more biomarkers may be determined via binding to one or more antibodies that are specific to the one or more biomarkers. Suitable antibodies are known or may be generated using known techniques.
  • Suitable methods for detecting antibody levels include, but are not limited to, immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, Western blotting and immunoprecipitation.
  • immunoassays such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, Western blotting and immunoprecipitation.
  • the level of the one or more biomarkers is determined using a sandwich immunoassay.
  • the antibody may be, for example, a monoclonal antibody, polyclonal antibody, multispecific antibody (e.g., bispecific antibody) or fragment thereof provided that it specifically binds to the biomarker being detected.
  • Antibodies may be obtained by standard techniques comprising immunising an animal with a target antigen and isolating the antibody from serum.
  • Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. (Kohler et al. (1975) Nature 256: 495) or may be made by recombinant DNA methods (e.g. disclosed in US 4816567).
  • Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (Clackson et al.
  • the level of the one or more biomarkers may be determined by staining the sample with a reagent that labels one or more of the biomarkers. "Staining" is typically a histological method, which renders the biomarker detectable, for example by microscopic techniques, such as those using visible or fluorescent light.
  • the biomarker is detected in the sample by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the biomarker may be detected by an antibody that binds specifically to one or more of the biomarkers.
  • direct and indirect assays Two general methods of antibody-based detection (including for IHC-based methods) are available: direct and indirect assays.
  • binding of antibody to the target antigen is determined directly.
  • This direct assay uses a labelled reagent, such as a fluorescent tag or an enzyme-labelled primary antibody, which can be visualised without further antibody interaction.
  • unconjugated primary antibody binds to the antigen and then a labelled secondary antibody binds to the primary antibody.
  • a chromogenic or fluorogenic substrate is added to provide visualisation of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used may be labelled with a detectable moiety.
  • Numerous labels are available, including radioisotopes, colloidal gold particles, fluorescent labels and various enzyme-substrate labels.
  • Fluorescent labels include, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin and phycocyanin, and/or derivatives of any one or more of the above.
  • the fluorescent labels can be conjugated to the antibody using known techniques.
  • the enzyme generally catalyses a chemical alteration of the chromogenic substrate that can be detected microscopically, for example under visible light.
  • the enzyme may catalyse a colour change in a substrate, or may alter the fluorescence or chemiluminescence of the substrate.
  • enzymatic labels include luciferases (e.g. firefly luciferase and bacterial luciferase; e.g.
  • IHC methods may comprise a step of detecting stained regions within an image. Pixels in the image corresponding to staining associated with the biomarker may be identified by colour transformation methods, for example as disclosed in US 6553135 and US 6404916. In such methods, stained objects of interest may be identified by recognising the distinctive colour associated with the stain.
  • the method may comprise transforming pixels of the image to a different colour space and applying a threshold value to suppress background staining. For example, a ratio of two of the RGB signal values may be formed to provide a means for discriminating colour information. A particular stain may be discriminated from background by the presence of a minimum value for a particular signal ratio. For example, pixels corresponding to a predominantly red stain may be identified by a ratio of red divided by blue (R/B) which is greater than a minimum value.
  • R/B ratio of red divided by blue
  • Kong et al. (Kong et al. (2013) Am. J. Clin. Nutr. 98: 1385-94) describes the use of the avidin- biotin-peroxidase method and two independent investigators counting the number of positively stained cells.
  • Detection using aptamers may comprise the following steps: aptamers that specifically recognise the biomarker may be synthesised using standard nucleic acid synthesis techniques or selected from a large random sequence pool, for example using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique; aptamers are mixed with the samples so that aptamer-protein complexes are formed; non-specific complexes are separated; bound aptamers are removed from their target proteins; aptamers are collected and measured, for example using microarrays or mass spectrometry techniques.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • Aptamers can be single stranded DNA or RNA sequences that fold into a unique 3D structure having a combination of stems, loops, quadruplexes, pseudoknots, bulges or hairpins.
  • the molecular recognition of aptamers results from intermolecular interactions, such as the stacking of aromatic rings, electrostatic and van der Waals interactions, or hydrogen bonding with a target compound.
  • the specific interaction between an aptamer and its target is complemented through an induced fit mechanism, which requires the aptamer to adopt a unique folded structure to its target.
  • Aptamers can be modified to be linked with labelling molecules such as dyes or immobilised on the surface of beads or substrates for different applications.
  • the invention comprises a step of determining the level of one or more biomarkers in one or more samples obtained from a subject.
  • the sample is a sample derived from blood.
  • the sample derived from blood may contain a blood fraction or may be whole blood.
  • the sample derived from blood is a plasma or serum sample, most preferably a serum sample.
  • the subjects disclosed herein are preferably mammals, particularly preferably humans. Both human and veterinary applications are within the scope of the invention.
  • the subject may be at risk of developing an impaired blood-brain barrier secondary to any of the following conditions: traumatic brain injury, hypoxic ischemia, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
  • the subject may be at risk of developing an impaired blood-brain barrier associated with vascular cognitive impairment, vascular dementia, age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
  • vascular cognitive impairment associated with vascular cognitive impairment, vascular dementia, age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • the subject may be, for example, an ageing human subject, such as a human over the age of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old.
  • the subject is a human over the age of 55 years old.
  • the age of the animal would be scaled from the human situation using the average lifespan for calibration.
  • references herein to treatment include curative, palliative and prophylactic treatment; although in the context of the invention references to preventing are more commonly associated with prophylactic treatment. Treatment may also include arresting progression in the severity of a disease.
  • dietary intervention refers to an external factor applied to a subject which causes a change in the subject's diet.
  • the dietary intervention is a diet supplemented with vitamins and/or minerals, preferably vitamin B.
  • the dietary intervention is a diet supplemented with omega-3 fatty acids.
  • the dietary intervention comprises increasing vitamin B intake by the subject, preferably by administering a vitamin B supplement.
  • the dietary intervention comprises increasing omega-3 fatty acid intake by the subject, preferably by administering an omega-3 fatty acid supplement.
  • the vitamin B may be, for example, vitamin B12, vitamin B6 and/or folic acid.
  • the omega-3 fatty acid may be, for example, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), preferably EPA.
  • the diet may be one which is adjusted to the starting body weight of the subject.
  • the dietary intervention may comprise administration of at least one diet product.
  • the diet product may be a meal replacement product or a supplement product.
  • the diet product may include food products, drinks, pet food products, food supplements, nutraceuticals, food additives or nutritional formulae.
  • the study participants with MCI and the participants with mild dementia have been recruited among outpatients with cognitive impairment referred to the Memory Clinics, Departments of Psychiatry, and the Leenaards Memory Center, Department of Clinical Neurosciences, University Hospitals of Lausanne for investigation of their cognitive complaints.
  • the diagnosis of MCI or of mild dementia was based on neuropsychological and clinical evaluation, and made by a consensus conference of psychiatrists and/or neurologists, and neuropsychologists prior to the inclusion in the study.
  • MCI criteria required memory impairment ( ⁇ 1.5 SD below the age, gender and education adjusted mean on the Buschke Double Memory Test verbal memory score) (Buschke, Sliwinski, Kuslansky, & Lipton, Neurology 1997, 48 (4), 989-997), and/or impairment in another cognitive domain such as executive tasks, and a Clinical Dementia Rating (CDR) (Morris, Neurology 1993, 43 (1 1 ), 2412-2414) equal to 0.5.
  • CDR Clinical Dementia Rating
  • the neuropsychological assessment includes measures of memory and other major cognitive domains such as language, attention and executive functioning. This assessment consists of the Mini Mental State Examination (Folstein MF et al. 1975, J. Psychiatr. Res 12, 189-198), the Buschke Double Memory Test (Buschke H et al. 1997, Neurology, 48, 989- 997) the digit span forward and backward (Wisdom NM et al. 2012, Arch Clin
  • the functional assessment includes the ADL and instrumental ADL (IADL) (Lawton MP et al. 1969, Gerontologist 9, 179-186), as well as the CDR (Morris JC 1993, Neurology, 43, 2412- 2414).
  • the neuropsychological test battery, ADL and IADL, and the CDR were used to verify inclusion and exclusion criteria.
  • CSF Cerebrospinal fluid
  • Venous and lumbar punctures were performed between 8:30 and 9:30 am in the Memory centres after an overnight fast. Blood was drawn into EDTA containing vacutainers (Sarstedt, Germany) and spun down to permit aliquots of supernatant (plasma and serum) for the analysis. Lumbar puncture and spinal fluid collection was performed on subjects in sitting or lying position with a 22G "atraumatical" spinal needle to capture 10-12 mL of CSF into polypropylene tubes. CSF cell count and protein quantification were performed in 2-3 mL and the remaining CSF was centrifuged, aliquoted, snap-frozen and stored at -80 °C until assay.
  • a "sandwich” immunoassay (Meso Scale Discovery (MSD), Rockville, MD, USA) quantified 37 analytes (IFN-gamma, IL-1 B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TNFa, IL-1 a, IL-5, IL-7, IL- 12/23p40, IL-15, IL-16, IL-17A, TNF-B, VEGFA, Eotaxin, MIP-1 B, Eotaxin-3, TARC, IP-10, MIP-1 a, MCP-1 , MDC, MCP-4, VEGF-C,VEGF-D, Tie-2, Flt-1 , PIGF, bFGF, SAA, CRP, VCAM-1 , ICAM-1 ) in serum.
  • analytes IFN-gamma, IL-1 B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, T
  • Samples were measured following the manufacturer's instructions. Briefly, the 96-well plates pre-coated with capture antibodies were blocked with 5% MSD Blocker A Solution. Calibrator dilutions were prepared and samples were diluted as recommended for each kit with MSD Diluents. Samples and calibrators were then added to the plates and incubated at room temperature with shaking for 2 h. Plates were washed three times with a home-prepared solution of 10 x phosphate-buffered saline (PBS), pH 7.4 (Corning, Manassas, VA, USA)- Tween 20 (Fisher Scientific, Pittsburgh, PA, USA).
  • PBS phosphate-buffered saline
  • pH 7.4 Corning, Manassas, VA, USA
  • Tween 20 Fisher Scientific, Pittsburgh, PA, USA.
  • MSD Read buffer was added and plates were read on an MSD instrument (SECTOR Imager 6000 reader). Data were generated and interpolated using MSD Discovery Workbench software.
  • Buffer exchange was performed with Strata-X 33u Polymeric reversed-phase (RP) (30mg/1 ml_) cartridges mounted on a 96-hole holder and a vacuum manifold, all from Phenomenex (Torrance, CA, USA). Samples were subsequently evaporated with a vacuum centrifuge (Thermo Scientific) and stored at -80 °C.
  • RP Polymeric reversed-phase
  • RP liquid chromatography tandem mass spectrometry RP liquid chromatography tandem mass spectrometry
  • RP-LC MS/MS was performed with hybrid linear ion trap-Orbitrap (LTQ-OT) Elite and an Ultimate 3000 RSLC nano system (Thermo Scientific).
  • Proteolytic peptides (injection of 5 ⁇ _ of sample) were trapped on an Acclaim PepMap 75 ⁇ ⁇ 2 cm (C18, 3 ⁇ , 100 A) pre-column and separated on an Acclaim PepMap RSLC 75 ⁇ ⁇ 50 cm (C18, 2 ⁇ , 100 A) column (Thermo Scientific) coupled to a stainless steel nanobore emitter (40 mm, OD 1/32") mounted on a Nanospray Flex Ion Source
  • the OT resolution was 120000 (ion population of 1 ⁇ 10 6 ) with an m/z window from 300 to 1500.
  • HCD collisional dissociation
  • a maximum of 10 precursor ions were selected for MS/MS.
  • Proteome Discoverer version 1 .4, Thermo Scientific was used as data analysis interface. Identification was performed against the human UniProtKB/Swiss-Prot database (08/12/2014 release) including the LACB sequence (20194 sequences in total). Mascot (version 2.4.2, Matrix Science, London, UK) was used. Variable amino acid modifications were oxidized methionine, deamidated asparagine/glutamine, and 6-plex TMT-labelled peptide amino terminus (+ 229.163 Da). 6-plex TMT-labelled lysine (+ 229.163 Da) was set as fixed modifications as well as carbamidomethylation of cysteine.
  • Trypsin was selected as the proteolytic enzyme, with a maximum of two potential missed cleavages.
  • Peptide and fragment ion tolerance were set to, respectively, 10 ppm and 0.02 Da.
  • All Mascot result files were loaded into Scaffold Q+S 4.4.1.1 (Proteome Software, Portland, OR, USA) to be further searched with X! Tandem (version CYCLONE (2010.12.01.1 )). Both peptide and protein FDRs were fixed at 1 % maximum, with a 2 unique peptide criterion to report protein identification.
  • Quantitative values were exported from Scaffold Q+S as log2 of the protein ratio fold changes with respect to their measurements in the biological reference, i.e., mean log2 values after isotopic purity correction but without normalization applied between samples and experiments.
  • each serum sample was thawed only once and homogenized for 10 s using a vortex mixer. Subsequently, the samples were diluted (1 :10) using the diluent solution.
  • a human serum pool was aliquoted and spiked with ICP standards. Three different concentration levels, adjusted to the concentration range of the respective elements in human serum were prepared.
  • spiked samples were prepared: set 1 consisting of level 1-3 (Mg, P, S, K, Ca and Mo) and set 2, level 1-3 (B, Al, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Cd, Sn, I, Cs, Ba Hg and Pb).
  • level 1-3 Mg, P, S, K, Ca and Mo
  • level 1-3 B, Al, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Cd, Sn, I, Cs, Ba Hg and Pb).
  • HTG Molecular Diagnostics, Inc. (Tucson, AZ) for miR profiling using qNPA.
  • the expression of 2,083 human miRs transcripts was analysed using HTG EdgeSeq miRNA Whole Transcriptome Assay and next-generation sequencing. The process consist of just five simples steps with low hands-on time.
  • Plasma sample 25 ⁇ are combined with 25 ⁇ of pre-warmed HTG Plasma Lysis Buffer and 1/10 Proteinase K and incubated for 3h at 50°c with orbital shaking. After sample preparation, they are processed using the automated HTG EdgeSeq system. Samples was hybridized with miRNA probes in qNPA reaction in which an excess of nuclease protection probes (NPPs) complimentary to each miRNA hybridize to their target.
  • NPPs nuclease protection probes
  • probes specific for the whole miRNA were incubated with the samples, forming specific probe-miRNA duplexes, then unhybridized probes were digested by S1 Nuclease, followed by alkaline hydrolysis to destroy the sample miRNA in the duplexes. This left intact probes with stoichiometric concentrations proportional to the abundance of specific miRNA in the original sample.
  • the second step of library preparation is the PCR amplification of capture probes with barcoded sequencing adapter.
  • Hemo KlenTaq® enzyme (NEB, M0332S) was used for plasma/serum sample.
  • the index adapter are provided by supplier: HTG EdgeSeq sequencing tag Pack.
  • the final library was purified with AMPure XP beads (Beckman Coulter, Brea, CA) in ratio x2.5. After quantification with Qubit system (Life Technologies) and profiling with LabChip DNA NGS 3K Assay (PerkinElmer, Inc), libraries were pooled equimolar at 2nM and by 24 samples for sequencing.
  • HTG EdgeSeq parser After sequencing, use the HTG EdgeSeq parser for post-sequencing data processing. Load the individual FastQ files generated by the sequencer to the parser. Probe sequences are aligned to the sequencing reads, making data processing simple and very quick.
  • the system generate an excel sheet with the count for each miRNA probes tested (2,083) and for positive and negative control.
  • the miRNA assay contains both positive and negative control probes. These probes are used to assess the performance of the chemistry to ensure quality data is produced. Raw count dataset were transfer to Quartz Bio S.A. for analysis.
  • Sample preparation was done with 50 ⁇ _ of blood plasma or serum with a mixture of 10 ⁇ _ of labelled internal standards and 140 ⁇ _ of ice-cold methanol (0.1 % formic acid) for protein precipitation were added. Samples were agitated by vortex (5 min) followed by centrifugation at 14500 rpm for 10 min at 4°C. The supernatant was then collected and subjected to derivatization.
  • AccQ-Tag Ultra Derivatization was performed using the AccQ-Tag Ultra Derivatization Kit Amino Acid Analysis (Waters Corp.) following manufacturer's instructions: 10 ⁇ _ of either a standard amino acid mix solution or the supernatant of the sample was mixed with 70 ⁇ _ of AccQ-Tag Ultra borate buffer (pH 8.8). The derivatization was carried out by adding 20 ⁇ _ of reconstituted AccQ-Tag Ultra reagent (3 mg mL-1 of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, or AQC in acetonitrile) to the buffered mixture. The sample was then immediately vortexed followed by incubation at 55°C for 10 min.
  • UPLC-MS/MS Ultra Performance Liquid Chromatography coupled to tandem mass spectrometry
  • UPLC-MS/MS analysis was performed on a Waters Acquity UPLC system coupled on-line to a Waters Xevo TQ mass spectrometer using an electrospray ionization (ESI) probe.
  • Chromatographic separation was achieved using a Waters AccQ-Tag Ultra column (100 x 2.1 mm x, 1 .7 ⁇ particle size) using a binary system of eluents for gradient elution.
  • Eluent A contained 10% of AccQ-Tag Ultra Eluent A concentrate (Waters Corp.) while eluent B was composed of 100% AccQ-Tag Ultra Eluent B (Waters Corp.).
  • Eluent B was composed of 100% AccQ-Tag Ultra Eluent B (Waters Corp.).
  • the autosampler temperature was set at 20 °C and the column temperature at 55°C.
  • the sample injection volume was 2 ⁇ _.
  • the UPLC-MS/MS system control and data acquisition were performed with the Waters Corporation MassLynxTM software. Data analysis was conducted with the TargetLynxTM software (Waters Corporation).
  • Lipid extraction was carried out in high grade polypropylene deep well plates. Fifty microliters of diluted plasma (50x) (equivalent of 1 mL of undiluted plasma) was mixed with 130 mL of ammonium bicarbonate solution and 810 mL of methyl tert-butyl ether/methanol (7:2, v/v) solution was added. Twenty-one microliters of internal standard mixture was pre-mixed with the organic solvents mixture.
  • the internal standard mixture contained: 50 pmol of lysophasphatidylglycerol (LPG) 17:1 ,50 pmol of lysophosphatic acid (LPA) 17:0, 500 pmol of phosphatidylcholine (PC) 17:0/17:0, 30 pmol of hexosylcer-amide (HexCer) 18:1 ;2/12:0, 50 pmol of phosphatidylserine(PS) 17:0/17:0, 50 pmol of phosphatidylglycerol (PG) 17:0/17:0, 50 pmol of phosphatic acid (PA) 17:0/17:0, 50 pmol of lysophposphatidylinositol (LPI 17:1 ), 50 pmol of lysophos-phatidylserine (LPS) 17:1 , 1 nmol of cholesterol (Choi) D6,
  • the plate was then sealed with a teflon-coated lid, shaken at 4°C for 15 min, and spun down (3000 g, 5 min) to facilitate separation of the liquid phases and clean-up of the upper organic phase.
  • Hundred microliters of the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. Dried lipids were re-suspended in 40 mL of 7.5 mM ammonium acetate in chloroform/methanol/propanol (1 :2:4, v/v/v) and the wells were sealed with an aluminum foil to avoid evaporation and contamination during infusion. All liquid handling steps were performed using Hamilton STARIet robotic platform with the AntiDroplet.
  • Samples were analysed by direct infusion in a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences, Ithaca, NY, USA). Five microliters were infused with gas pressure and voltage set to 1 .25 psi and 0.95 kV, respectively. The delivery time was set to 4 min and 55 s with contact closure delay of 20 s to avoid initial spray instability. Polarity switch from positive to negative mode was set at 135 s after contact closure. Samples were analysed in both polarities in a single acquisition.
  • the MS acquisition method starts with positive ion mode by acquiring the m/z 402-412 in MS positive mode at resolution of 140000 to monitor the [Choi + NH 4 + ] + ion for 12 s. All individual scans in every segment are the average of 2 micro-scans.
  • Automatic gain control AGC was set to 5 x 10 5 and maximum ion injection time was set to 200 ms.
  • AGC was set to 10 6 and maximum ion injection time was set to 50 ms.
  • MS/MS resolution of 17500
  • the inclusion list contains all the masses from 500.5 to 999.75 with 1 Da intervals.
  • AGC was set to 10 5 and maximum ion injection time was set to 64 ms.
  • the isolation width was set to 1.0 Da, first mass of MS/MS acquisition was 250 Da and normalized collision energy was set to 20%.
  • Both MS and MS/MS data are combined to monitor SE, DAG, and TAG ions as ammonium adducts. After polarity switch to negative ion mode, a lag of 15 s before acquisition was inserted to allow spray stabilization.
  • MS/MS Resolution of 17500
  • This inclusion list contains all the masses from 590.5 to 939.5 with 1 Da intervals.
  • AGC was set to 10 5 and maximum ion injection time was set to 64 ms.
  • Isolation width was set to 1 .0 Da, first mass of MS/MS acquisition was 150 Da, and normalized collision energy was set to 35%.
  • Both MS and MS/MS data were combined in order to monitor PC, PC 0-, HexCer, Cer, SM as acetate adducts and PS, PG, PA, PE, PE 0-, and PI as deprotonated anions.
  • DNA was extracted from whole blood using the QIAsymphony DSP DNA Kit (Qiagen, Hombrechtikon, Switzerland).
  • the SNV rs429358 and rs7412 were genotyped using the Taqman assays C_3084793_20 and C_904973_10 respectively (Thermo Fischer Scientific, Waltham, MA USA).
  • Biomarker data was quality-controlled prior to hypothesis testing by first excluding those with more than 5% missing data. The remaining missing data ( ⁇ 5%) was imputed by randomly drawing a measure between the observed range of biomarker values. Biomarker data was then log-transformed to approach a Gaussian distribution, and standardised prior to final hypothesis testing.
  • the association of BBB impairment with demographic variables age, gender, education, ApoE4 presence, CSF Abeta, CSF p-tau, CSF tau, CDR category, and status in terms of diabetes, hypertension, and hypercholesterolemia was analysed using logistic regression models.
  • the performance of the obtained classifier was assessed by measuring (i) its area under the Receiver Operating Characteristic (ROC) curve and its 95% confidence interval (using a bootstrap approach with 1000 iterations) and (ii) its accuracy (cumulated proportion of true-positives and true-negatives in the obtained 2x2 confusion matrix). The accuracy of this model was 86.4% with an AUC of 0.75.
  • ROC Receiver Operating Characteristic
  • BBB impairment was defined as CSF-to-serum ratio of albumin greater than 9.0.
  • the best predictive model takes into account the 4 miRNA and other 7 biomarkers.
  • the miRNA biomarkers were miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, together with other blood-based biomarkers phosphatidylinositol-glycan-specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , peroxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp) have been determined as biomarkers for identifying BBB impairment or risk of developing BBB impairment.
  • the accuracy of this model was 93.0% with an AUC if 0.96.
  • LASSO Least absolute shrinkage and selection operator logistic regression selected biomarkers that best predict BBB impairment.
  • a reference model was initially generated, testing variables that are likely to be available to the clinicians to provide a benchmark for comparison with the models that included blood biomarkers.
  • a 10-fold cross- validation process was performed for each LASSO analysis, which allows estimating the confidence interval of the misclassification error for each value of the regularization parameter ⁇ .
  • the LASSO analyses were repeated 100 times (1000 times for the reference model).
  • the models that minimized the upper limit of the cross-validated misclassification error confidence interval across the 100 runs with less than 20 features were selected. Their performance was assessed by receiver operating characteristic (ROC) area under the curve (AUC) estimation using a bootstrap approach with 1000 iterations. Results were compared visually and formally tested for significance against the reference model using ROC AUC and accuracy using a McNemar test.
  • ROC receiver operating characteristic
  • AUC area under the curve

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Abstract

A method for determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) comprising determining the level of one or more biomarkers in one or more samples obtained from the subject, wherein the one or more biomarkers comprise a micro-RNA.

Description

MICRO-RNA BIOMARKERS OF BLOOD-BRAIN BARRIER DYSFUNCTION FIELD OF THE INVENTION
The present invention relates to biomarkers and biomarker combinations that may be used to determine whether a subject has or is at risk of developing an impaired blood-brain barrier (BBB). The invention also relates to methods for identifying individuals with an impaired blood brain barrier or at risk of developing an impaired blood brain barrier concomitant with disease or other conditions and it also provides methods of treatment or prevention of blood brain barrier impairment in said individuals.
BACKGROUND TO THE INVENTION The blood-brain barrier (BBB) is a selective barrier that separates circulating blood from the brain. The BBB is comprised of endothelial cells bound together by tight junction proteins that form the blood facing side of the lumen of the small cerebral blood vessels. In addition, astrocytes, in particular, projections from those cells termed astrocytic feet and pericytes contribute to the structure and function of the BBB. The endothelial cells of the BBB express multiple substrate-specific transport systems that control the transport of nutrients, energy metabolites, and other essential molecules from the blood into the brain and the transport of metabolic waste products from the brains interstitial fluid into the blood (Aspelund A. et al. 2015, J. Exp. Med 212, 991 -999). As such, the BBB serves as a key homeostatic site of the nervous system since it connects the central nervous system (CNS) systemic circulation, and major systems in the body such as respiratory, renal, hepatic and immune systems (Zhao,Z et al. 2015, Cell 163, 1064-1078).
BBB dysfunction is considered to be a vascular contributor to the risk for the development of age-related cognitive decline, cognitive impairment and dementia, including Alzheimer's disease and its progression. Imaging tools such as magnetic resonance imaging (MRI), computed tomography (CT) or positron emission tomography (PET) are currently used to identify potential dysfunction of the BBB (van der Haar et al. 2015, 49, 71 -81 ). The clinical measurement of cerebrospinal fluid to plasma albumin index is another means for identifying BBB dysfunction, however, it involves invasive sampling of the cerebrospinal fluid. Therefore, there is a need for better, minimally invasive, simplified methods of identifying dysfunctions of the BBB at an early stage.
In particular, there exists a significant need for methods of identifying BBB dysfunction in living subjects, in particular in subjects that do not exhibit symptoms of or have not been diagnosed with a cognitive impairment. Early diagnosis of subjects with an impaired BBB may enable therapeutic intervention, which may prevent or reduce the risk of the subject developing conditions associated with an impaired BBB, for example, cognitive impairments such as vascular cognitive impairment, vascular dementia, mild cognitive impairment (MCI), age- related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
SUMMARY OF THE INVENTION
The inventors have demonstrated that certain biomarkers can identify subjects, with blood- brain barrier (BBB) impairment. In particular, certain microRNAs (miRNAs) can be used as biomarkers alone or together with other biomarkers to identify BBB impairment. MicroRNAs (miRNAs) are small, non-coding RNAs around 17-25 nucleotides in length. They are regulatory RNA molecules that function to regulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes. Deregulation of miRNA expression has been shown to have an impact on health and diseases (Wang et al. 2016, J. Cell Phys. 231 :25-30). miRNAs are differentially expressed in different cell types, tissues and fluids. They are considered to be an excellent class of blood-based biomarkers since their expression is highly stable in plasma and serum and they have the advantage over measurement of cerebrospinal fluid in that there are relatively easy to monitor.
Specifically, the inventors collected cerebrospinal fluid (CSF) and blood serum samples from 1 18 adults aged 55 and older to analyse the cross-sectional relationship between blood-based biomarkers and BBB function. BBB dysfunction was defined a priori as a CSF-to-serum albumin ratio greater than or equal to 9.0. The concentration of albumin found in the CSF divided by the concentration of serum albumin. The amount of albumin crossing the healthy BBB is strictly regularized. Therefore, an increased albumin ratio is considered to be a measure of BBB impairment.
The inventors carried out Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression analysis to select the biomarkers that best classified subjects with BBB impairment. Subsequently, diagnostic accuracy was assessed by calculating area under the receiver operating characteristic (ROC) curve. The inventors determined biomarkers for identifying BBB impairment.
In one aspect of the invention, miRNA biomarkers have been determined as biomarkers for identifying BBB impairment. In another aspect of the invention, a miRNA profile of biomarkers has been identified in Table 1 for identifying BBB impairment. In one embodiment, the measurement of a particular miRNA biomarker is compared to a control to identify BBB impairment.
In another aspect of the invention, miRNA biomarkers such as miR-204-5p, miR-501 -5p, miR- 136-3p, miR-34a-5p, have been determined as biomarkers for identifying BBB impairment.
In a further aspect of the invention, miRNA biomarkers such as miR-204-5p, miR-501 -5p, miR- 136-3p, miR-34a-5p, together with other blood-based biomarkers such as phosphatidylinositol-glycan-specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , peroxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp) have been determined as biomarkers for identifying BBB impairment.
Accordingly, in one aspect, the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
In another aspect, the invention provides a method of identifying a subject with impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein the at least one miRNA is selected from the group of miRNAs comprising Table 1 and an increased or decreased level of expression of miRNA in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
In another aspect, the invention provides a method of identifying a subject with impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein the at least one miRNA is selected from the group of miRNAs consisting of: miR 204- 5p, miR 501 -5p, miR136-3p or miR34a-5p and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB). In another aspect, the invention provides a method of identifying a subject with impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein the at least one miRNA is selected from the group of miRNAs consisting of: miR 204- 5p or miR 501 -5p and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
In a further aspect, the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNA in a reference wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
In another aspect, the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNAs in a reference wherein the at least two miRNAs are selected from the group of miRNAs comprising Table 1 and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB). In another aspect, the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNA in a reference wherein the at least two miRNAs are selected from the group of miRNAs consisting of miR204-
5p, miR 501 -5p, miR136-3p or miRNA 34a-5p and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
In another aspect, the invention provides a method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNA in a reference wherein the at least two miRNAs are miR204-5p and miR501 -5p and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB). In yet another aspect, the invention provides a method of identifying a subject with an impaired blood-brain barrier with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) wherein in addition to miRNA biomarkers further biomarkers are measured.
In one aspect, the invention provides a method of identifying a subject with an impaired blood- brain barrier or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of miRNA in the biological sample from the subject c) comparing the level of expression of miRNA in the sample to a level of expression of the miRNA in a reference d) further measuring additional biomarkers wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB). In another aspect, the invention provides a method of identifying a subject with an impaired blood brain barrier or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of miRNA in the biological sample from the subject c) comparing the level of expression of miRNA in the sample to a level of expression of the miRNA in a reference d) further measuring additional biomarkers wherein the additional biomarkers selected from the group consisting of: PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, and tryptophan in one or more samples obtained from the subject and an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
In a further aspect, the invention provides a blood-brain barrier (BBB) impairment score (S) using the formula: S = A x (miR-204-5p) + B x (miR-501 -5p) + C x (miR-136-3p) + D x (miR-34a-5p) + E x (PHLD) + F x (strontium) + G (PRG4) + H (cholestrol ester 17:1 ) + I x (PRDX2) + J x (IL16) + K x (tryptophan) wherein A, B, C, D, E, F, and G, H, I, J and K are coefficients.
In one aspect, the invention provides that the reference is a sample from a normal healthy subject without blood brain barrier impairment.
In another aspect, the invention provides that the reference is a sample from the same subject at a different time point.
In another aspect, the invention provides that the biological sample is selected from the group consisting of a bodily fluid, tissue or cell. In another aspect, the invention provides that the bodily fluid sample is a blood sample.
In another aspect, the invention provides that the bodily fluid is a serum sample.
In one aspect, the invention provides the identification of a subject with an impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) is associated with vascular cognitive impairment, vascular dementia, age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
In another aspect, the invention provides the identification of a subject with an impaired blood- brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) is associated with any of the following conditions: traumatic brain injury, hypoxic ischemia, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
In one aspect, the invention provides a method of treating or preventing blood-brain barrier (BBB) impairment comprising the steps: a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) by measuring biomarkers of the invention according to the method of the invention described above b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof. In one embodiment, the intervention is a dietary intervention.
In one aspect, the invention provides a method of treating or preventing blood-brain barrier (BBB) impairment comprising the steps: a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) according to the method of identifying the level of expression of the biomarkers of the invention; and b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof wherein the dietary intervention comprises increasing vitamin B intake by the subject, preferably by administering a vitamin B supplement.
In another aspect, the invention provides a method of treating or preventing blood brain barrier (BBB) impairment comprising the steps: a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) according to the method of identifying the level of expression of the biomarkers of the invention; and b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof wherein the dietary intervention comprises increasing omega-3 fatty acid intake by the subject, preferably by administering an omega-3 fatty acid supplement.
In one embodiment, the miRNA is mammalian miRNA.
In a preferred embodiment, the miRNA is human miRNA (hsa-miRNA). In one embodiment, the miRNA is selected from Table 1 .
In another embodiment, the miRNA is selected from miR-204-5p, miR-501 -5p, miR-136-3p, or miR-34a-5p.
In a preferred embodiment, the miRNA is miR-204-5p and/or miR-501 -5p.
In one embodiment, miR-204-5p is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In another embodiment, miR-501 -5p is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB). In another embodiment, miR-136-3p is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In yet another embodiment, miR-34A-5p is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In one embodiment, the method further comprises determining the level of another blood- based biomarker in a sample from the subject.
In one embodiment, the method further comprises determining the level of one or more biomarkers selected from the group consisting of: PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, or tryptophan in one or more samples obtained from the subject.
In one embodiment, PHLD is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB). In another embodiment, strontium is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In another embodiment, PRG4 is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In another embodiment, cholesterol ester 17:1 is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood- brain barrier (BBB). In another embodiment, PRDX2 is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In another embodiment, IL-16 is increased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In yet another embodiment, tryptophan is decreased with respect to a reference if the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB).
In one embodiment, the method comprises determining the level of miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, and tryptophan in one or more samples obtained from the subject.
In one embodiment, the level of the one or more biomarkers is compared with one or more reference values. In this case, preferably each biomarker level in each sample and the corresponding reference values are determined using the same analytical method. The reference values may be based on values (e.g., averages) of the one or more biomarkers in populations of subjects who have, for example, previously been identified as having normal or impaired blood-brain barriers.
In one embodiment, the method comprises determining a value that represents the prediction of blood-brain barrier impairment (BBB). This may be termed a blood-brain barrier impairment score (S) and may be calculated using the formula: S = A x (miR-204-5p) + B x (miR-501 -5p) + C x (miR-136-3p) + D x (miR-34a-5p) + E x (PHLD) + F x (strontium) + G (PRG4) + H (cholesterol ester 17:1 ) + I x (PRDX2) + J x (IL16) + K x (tryptophan) wherein A, B, C, D, E, F, G, H, I, J and K are coefficients. The coefficients may be chosen based on a pre-determined model. Blood-brain barrier impairment may be predicted if S is above or below a pre-determined level, for example if S > 0.
In one embodiment, the method comprises determining a blood-brain barrier impairment score (S) using the formula:
S = A x logio(miR-204-5p) + B x logio(miR-501 -5p) + C x logio(miR-136-3p) + D x logio(miR-34a-5p) + E x log2(PHLD) + F x logio(strontium) + G x log2(PRG4) + H x logio(cholesterol ester 17:1 ) + I x log2(PRDX2) + J x logio(IL16) + K x logio(tryptophan) wherein blood brain barrier impairment is predicted if S > 0; wherein miR-204-5p, miR-501 -5p, miR-136-3p, and miR-34a-5p in plasma are given as counts; PHLD, PRG4 and PRDX2 in plasma are given as fold change ratios; strontium in serum is given in ng-mL"1; cholesterol ester in plasma is given as signal intensities; IL16 in serum is given in pg-mL"1; and tryptophan in plasma is given in μΜ.
In one embodiment, the level of miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, PHLD, strontium, PRG4, cholesterol ester, PRDX2, IL-16, and tryptophan are determined in a plasma or serum sample from a subject. In one embodiment, the levels of the one or more biomarkers are determined in one or more plasma or serum samples.
In one embodiment, the subject is a human.
In one embodiment, the subject is an ageing human. In another embodiment, the subject is a human over the age of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old. Preferably, the subject is a human over the age of 55 years old.
In one embodiment, the subject substantially does not exhibit any symptoms of a cognitive impairment.
In one embodiment, the subject has not been diagnosed with a cognitive impairment.
Preferably, the method is an in vitro method. In another aspect, the invention provides a kit for determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB), wherein the kit comprises one or more biomarker of the invention as disclosed herein.
In another aspect, the invention provides a kit for determining whether a subject is at risk of developing a cognitive impairment, wherein the kit comprises two or more biomarkers of the invention as disclosed herein.
In another aspect, the invention provides a method of treating or preventing blood-brain barrier (BBB) impairment comprising the steps:
(a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention; and
(b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof.
In another aspect, the invention provides a method of preventing or reducing the risk of a cognitive impairment comprising the steps:
(a) determining whether a subject is at risk of developing a cognitive impairment according to the method of the invention; and
(b) applying an intervention capable of preventing or reducing the risk of a cognitive impairment to a subject identified to be in need thereof. In one embodiment, the intervention is a dietary intervention.
In one embodiment, the dietary intervention comprises increasing vitamin B intake by the subject, preferably by administering a vitamin B supplement.
In one embodiment, the dietary intervention comprises increasing omega-3 fatty acid intake by the subject, preferably by administering an omega-3 fatty acid supplement. In another aspect, the invention provides a method of selecting a modification in lifestyle of a subject comprising the steps:
(a) determining whether the subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention; and (b) selecting a modification in lifestyle capable of improving blood-brain barrier (BBB) function in a subject identified to be in need thereof.
In another aspect, the invention provides a method of selecting a modification in lifestyle of a subject comprising the steps: (a) determining whether the subject is at risk of developing a cognitive impairment according to the method of the invention; and
(b) selecting a modification in lifestyle capable of preventing or reducing the risk of a cognitive impairment in a subject identified to be in need thereof.
In one embodiment, the method further comprises applying the selected modification in lifestyle to the subject.
In one embodiment, the modification in lifestyle comprises a dietary intervention as disclosed herein.
In another aspect, the invention provides a diet product for use in treating or preventing blood- brain barrier (BBB) impairment, wherein the diet product is administered to a subject determined to have an impaired blood-brain barrier or to be at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention.
In another aspect, the invention provides a diet product for use in preventing or reducing the risk of a cognitive impairment, wherein the diet product is administered to a subject determined to be at risk of developing a cognitive impairment according to the method of the invention. In another aspect, the invention provides the use of a diet product for the manufacture of a medicament for treating or preventing blood-brain barrier (BBB) impairment, wherein the diet product is administered to a subject determined to have an impaired blood-brain barrier (BBB) or to be at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention. In another aspect, the invention provides the use of a diet product for the manufacture of a medicament for preventing or reducing the risk of a cognitive impairment, wherein the diet product is administered to a subject determined to be at risk of developing a cognitive impairment according to the method of the invention.
In another aspect, the invention provides the use of a diet product for treating or preventing blood-brain barrier (BBB) impairment, wherein the diet product is administered to a subject determined to have an impaired blood-brain barrier (BBB) or to be at risk of developing an impaired blood-brain barrier (BBB) according to the method of the invention.
In another aspect, the invention provides the use of a diet product for preventing or reducing the risk of a cognitive impairment, wherein the diet product is administered to a subject determined to be at risk of developing a cognitive impairment according to the method of the invention.
In one embodiment, the diet product is a vitamin B supplement. In another embodiment, the diet product is an omega-3 fatty acid supplement. In another embodiment, the diet product is a folic acid supplement. In another aspect, the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood- brain barrier (BBB) according to the method disclosed herein.
In another aspect, the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject is at risk of developing a cognitive impairment according to the method disclosed herein.
In another aspect, the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood- brain barrier (BBB) given the levels of one or more biomarkers from the user, wherein the biomarkers are selected from the one or more biomarkers as disclosed herein.
In another aspect, the invention provides a computer program product comprising computer implementable instructions for causing a programmable computer to determine whether a subject is at risk of developing a cognitive impairment given the levels of one or more biomarkers from the user, wherein the biomarkers are selected from the one or more biomarkers as disclosed herein.
DESCRIPTION OF THE FIGURES
Figure 1 shows the box plot of the biomarker miR-204-5p comparing normal versus impaired blood-brain barrier function where there is a decrease in miR-204-5p in subjects with impaired blood-brain barrier function. Figure 2 shows the box plot of the biomarker miR-501 -5p comparing normal versus impaired blood-brain barrier function where there is a decrease in miR-501 -5p in subjects with impaired blood-brain barrier function.
Figure 3 shows the box plot of the biomarker miR-136-3p comparing normal versus impaired blood-brain barrier function where there is a decrease in miR-136-3p in subjects with impaired blood-brain barrier function.
Figure 4 shows the box plot of the biomarker miR-34a-5p comparing normal versus impaired blood-brain barrier function where there is an increase in miR-34a-5p in subjects with impaired blood-brain barrier function. Figure 5 shows the box plot of the biomarker PHLD comparing normal versus impaired blood- brain barrier function where there is an increase in PHLD in subjects with impaired blood-brain barrier function.
Figure 6 shows the box plot of the biomarker strontium comparing normal versus impaired blood-brain barrier function where there is a decrease in strontium in subjects with impaired blood-brain barrier function.
Figure 7 shows the box plot of the biomarker PRG4 comparing normal versus impaired blood- brain barrier function where there is an increase in PRG4in subjects with impaired blood-brain barrier function.
Figure 8 shows the box plot of the biomarker cholesterol ester 17:1 comparing normal versus impaired blood-brain barrier function where there is a decrease in cholesterol ester 17:1 in subjects with impaired blood-brain barrier function.
Figure 9 shows the box plot of the biomarker PRDX2 comparing normal versus impaired blood-brain barrier function where there is an increase in PRDX2 in subjects with impaired blood-brain barrier function. Figure 10 shows the box plot of the biomarker IL-16 comparing normal versus impaired blood- brain barrier function where there is an increase in IL-16 in subjects with impaired blood-brain barrier function.
Figure 1 1 shows the box plot of the biomarker tryptophan comparing normal versus impaired blood-brain barrier function where there is a decrease in tryptophan in subjects with impaired blood-brain barrier function. Receiver operating characteristic (ROC) curves for diagnosis of blood-brain barrier (BBB) impairment for Reference (labelled "REF ref") and Best models (labelled "meta"). For the Reference model the area under the curve (AUC) is 0.75 whereas for the Best model the area under the curve (AUC) is 0.96. The variables selected in the Best model are: the 4 miRNAs miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, and the other blood-based biomarkers PHLD, strontium, PRG4, cholesterol ester 17:1 , PRDX2, IL-16, and tryptophan.
DETAILED DESCRIPTION OF THE INVENTION
Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.
Blood-brain barrier (BBB)
The blood-brain barrier (BBB) is a selective barrier that separates circulating blood from the brain. The BBB is comprised of a monolayer of endothelial cells bonded by tight junction proteins that form the small cerebral blood vessel lumen. In addition, astrocytes (in particular, projections from those cells termed astrocytic feet) and pericytes contribute to the structure and function of the BBB.
The BBB governs entry of all peripherally circulating factors such as water diffusion, some gases and lipid-soluble molecules, and selective transport of other substances, such as glucose, amino acids, and micronutrients that are crucial to neuronal function. Conversely, the BBB protects the brain from the passage of toxic substances that may place the central nervous system (CNS) at risk. The term "impaired blood-brain barrier (BBB)" refers to a BBB that is not functioning correctly as a selective barrier between circulation and the brain. As used herein, the term "impaired blood-brain barrier (BBB)" may be equated with "dysfunctional blood-brain barrier (BBB)".
One example, is the case where certain larger proteins that are more abundant in circulation begin to penetrate the BBB ("leak") and infiltrate the cerebrospinal fluid (CSF) would be a case of impaired BBB.
An impaired BBB may occur, for example, in a subject having a higher than normal CSF-to- serum albumin ratio, for example a CSF-to-serum albumin ratio greater than or equal to 5, 6, 7, 8, or 9, preferably greater than or equal to 9. Cognitive impairment
Early diagnosis of subjects with an impaired BBB may enable therapeutic intervention, which may prevent or reduce the risk of the subject developing conditions associated with an impaired BBB, for example, cognitive impairments such as vascular cognitive impairment, vascular dementia, mild cognitive impairment (MCI), age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
For example, post-mortem analyses have demonstrated BBB damage in Alzheimer's disease patients. In addition, neuroimaging studies have shown the accumulation of iron and microbleeds in Alzheimer's disease patients, which suggests subtle haemorrhage or rupture of small vessels in the brain at some point across the lifespan. Further studies have shown that cerebrospinal fluid (CSF)-to-serum ratios of blood-derived albumin are higher in all dementia patients (including those suffering from Alzheimer's disease) when compared against age-matched controls. Indeed, this measure of BBB function associates with accelerated Alzheimer's disease progression independent of age and other Alzheimer's disease risk factors. BBB dysfunction may also present together with conditions of traumatic brain injury, hypoxic ischemia, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
BBB dysfunction therefore appears to be a significant risk factor for the development of cognitive impairments and their progression.
The term "cognition" refers to the set of mental thinking abilities and domains of attention and processing speed, short and long term memory, working memory, executive functions of planning and flexibility, decision making, judgment and evaluation, reasoning and "computation", problem solving, comprehension and language. "Cognitive impairment" refers to a deterioration in one or more these domains of cognition.
Levels of and improvements in cognition can be readily assessed by the skilled person using any of a number of validated neuropsychological tests standardized to assess, for example, speed of information processing, executive function and memory.
Suitable example tests include Mini Mental State Examination (MMSE), Clinical Dementia Rating (CDR), Cambridge Neuropsychological Test Automated Battery (CANTAB), Alzheimer's Disease Assessment Scale-cognitive test (ADAScog), Wisconsin Card Sorting Test, Verbal and Figural Fluency Test and Trail Making Test.
In addition, medical imaging of the brain provides an assessment of brain function. Examples of medical imaging techniques used for assessment of brain function include electroencephalography (EEG), magnetoencephalography (MEG), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Magnetic Resonance Imaging (MRI), functional Magnetic Resonance Imaging (fMRI), computerised tomography and long-term potentiation. Dynamic gadolinium enhanced MRI can also be used to assess blood brain barrier (BBB) function.
EEG, a measure of electrical activity of the brain, is accomplished by placing electrodes on the scalp at various landmarks and recording greatly amplified brain signals. MEG is similar to EEG in that it measures the magnetic fields that are linked to electrical fields. MEG is used to measure spontaneous brain activity, including synchronous waves in the nervous system.
PET provides a measure of oxygen utilisation and glucose metabolism. In this technique, a radioactive positron-emitting tracer is administered, and tracer uptake by the brain is correlated with brain activity. These tracers emit gamma rays which are detected by sensors surrounding the head, resulting in a 3D map of brain activation. As soon as the tracer is taken up by the brain, the detected radioactivity occurs as a function of regional cerebral blood flow. During activation, an increase in cerebral blood flow and neuronal glucose metabolism can be detected within seconds.
Suitable analysis can also be based on neuropsychological testing, general and neurological examinations and individual complaints of cognitive decline (e.g., subjective memory loss).
Cognitive impairment may be, for example, interpreted as a statistically significant difference in performance at any time point in a suitable test. Vascular dementia
Vascular dementia results from reduced blood flow to the brain, which damages brain cells. The reduced blood flow can occur for a number of reasons, including narrowing of the blood vessels in the brain (subcortical vascular dementia), stroke (single-infarct dementia) and numerous small strokes (multi-infarct dementia). The reduced blood flow may additionally be caused by Alzheimer's disease, a combination referred to as mixed dementia.
Early symptoms of vascular dementia include slowness of thought, difficulty with planning, difficulty with language, problems with attention and concentration, and behavioural changes. The symptoms typically worsen in steps, with intervening stable periods of months or years. Age-related cognitive decline
Age-related cognitive decline is the normal, non-pathological reduction in cognitive function that is associated with ageing. Although certain mental functions exhibit little age-related decline (e.g., language, reading and vocabulary skills, some numerical abilities and general knowledge) others decline from middle age (e.g., episodic memory, executive functions, speed of processing and reasoning). The extent to which subjects are affected by age-related cognitive decline varies between individuals.
Age-related cognitive decline usually is not considered severe enough to meet criteria for mild- cognitive impairment. Mild cognitive impairment (MCI) is considered to be objective assessment of cognitive deficit in at least one cognitive domain (age and gender adjusted) that does not impair activities of daily living. In contrast, probable Alzheimer's disease diagnosis requires impairment in at least two cognitive domains and impairment of activities of daily living.
Alzheimer's disease (AD)
Alzheimer's disease is caused by atrophy of areas of the brain. Although it is not known what initiates the atrophy, studies have found amyloid plaques, neurofibrillary tangles and acetylcholine imbalances in the brains of Alzheimer's patients. Vascular damage in the brain, which may damage healthy neurons, is also common in Alzheimer's patients.
Alzheimer's disease is a progressive condition that affects multiple brain functions. Early signs of the disease usually include minor memory problems, for example forgetting recent events or the names of places and objects. As the disease progresses, memory problems become more severe and additional symptoms can develop, such as confusion, disorientation, difficulty making decisions, problems with speech and language, and personality changes. Parkinson's disease (PD)
Parkinson's disease is a condition in which nerve cells in the substantia nigra become progressively damaged. Nerve cells in this area of the brain produce dopamine, which acts as a messenger between the parts of the brain and nervous system that control body movement. Damage to these nerve cells results in a reduction in the amount of dopamine produced in the brain, which has the effect of reducing function in the part of the brain controlling movement.
Symptoms of the Parkinson's disease include tremors, slow movement, and stiff and inflexible muscles. Parkinson's disease patients may also experience additional symptoms, including depression, constipation, insomnia, anosmia and memory problems. Other Conditions associated with BBB Impairment
Traumatic brain injury (TBI) may be another condition associated with BBB impairment. TBI is a non-congenital insult to the brain from an external mechanical force, possibly leading to permanent or temporary impairment of cognitive, physical, and psychosocial functions, with an associated diminished or altered state of consciousness. In addition, impairment of the blood-brain barrier (BBB) may be associated with any of the following conditions: hypoxic ischemia, transient ischemic attack, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies. Biomarkers microRNAs
The prefix "miR" is followed by a dash and a number, the latter often indicating order of naming. A capitalized "miR-" refers to the mature form of the miRNA, while the uncapitalized "miR-" refers to the pre-miRNA and the pri-miRNA, and "MIR" refers to the gene that encodes them. When two mature microRNAs originate from opposite arms of the same pre-miRNA and are found in roughly similar amounts, they are denoted with a -3p or -5p suffix. However, the mature microRNA found from one arm of the hairpin is usually much more abundant than that found from the other arm in which case, an asterisk following the name indicates the mature species found at low levels from the opposite arm of a hairpin. miRNA sequences have been deposited in miRBase database (http://www.mirbase.org/). The miRBase database is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download. miRBase is managed by the Griffiths-Jones lab at the Faculty of Life Sciences, University of Manchester with funding from the BBSRC. miRBase was previously hosted and supported by the Wellcome Trust Sanger Institute.
MicroRNAs are isolated from a body fluid.
In one embodiment, the miRNA is isolated from blood, plasma, serum, cerebrospinal fluid, urine, saliva, tears, amniotic fluid, colostrum, breast milk, bronchial lavage, peritoneal fluid, pleural fluid, or seminal fluid. In one embodiment, the miRNA is isolated from blood, preferably plasma.
In another embodiment, the miRNA is human.
Table 1 represents the miRNA measured in blood serum:
Table 1 - miRNA Measured in Serum
Figure imgf000022_0001
miR- >hsa-miR-34a-5p
SEQ ID ΜΙΜΆΤ0000255 34A-5p No.4 0.2697 1 .05 Up UGGCAGUGUCUUAGCUGGUUGU miR- >hsa-miR-6513-3p
SEQ ID MIMAT0025483
6513- No, 5
UCAAGUGUCAUCUGUCCCUAG
0,0798 1 ,05 Up
3p miR- >hsa-miR-624-5p
SEQ ID MIMAT0003293 624-5p No.6 0.2706 1 .04 Up UAGUACCAGUACCUUGUGUUCA miR- >hsa-miR-33a-3p
SEQ ID MIMAT0004506 33A-3p No,7 0.2820 1 ,04 Up CAAUGUUUCCACAGUGCAUCAC miR- >hsa-miR-212 -3p
S EC3 ID MIMAT0000269 212-3p No,8 0.2139 0.97 Down UAACAGUCUCCAGUCACGGCC miR- >hsa-miR-6734-5p
SEQ ID MIMAT0027369
6734- No.9
UUGAGGGGAGAAUGAGGUGGAGA
0,0270 0.93 Down
5p miR- >hsa-miR-6723-5p
SEQ ID MIMAT0025855
6723- No.10
AUAGUCCGAGUAACGUCGGGGC
0.0180 0.88 Down
5p miR- >hsa-miR-3180-3p
SEQ ID MIMAT0015058
3180- No.1 1
UGGGGCGGAGCUUCCGGAGGCC
0.1953 0.97 Down
3p miR- >hsa-miR-331-5p
SEQ ID MIMAT0004700 331 -5p No.12 0.0740 0,95 Down CUAGGUAUGGUCCCAGGGAUCC miR- >hsa-miR-6747-3p
SEQ ID MIMAT0027395
6747- No.13
UCCUGCCUUCCUCUGCACCAG
0,0403 0.94 Down
3p miR- >hsa-miR-4798-5p
S EG I D MIMAT0019974
4798- No.14
UUCGGUAUACUUUGUGAAUUGG
0.0462 0,94 Down
5p miR- >hsa-miR-5010-5p
SEQ ID MIMAT0021043
5010- No.15
AGGGGGAUGGCAGAGCAAAAUU
0.1874 0.97 Down
5p miR- >hsa-miR-500a-5p
SEQ ID MIMAT0004773
500A- No, 16 UAAUCCUUGCUACCUGGGUGAGA
0.2178 0.97 Down
5p miR- >hsa-miR-5010-3p
SEQ ID MIMAT0021044
5010- No, 17
UUUUGUGUCUCCCAUUCCCCAG
0.3201 0.97 Down
3p miR- >hsa-miR-4324
SEQ ID MIMAT0016876
4324 No.18 0.1987 0,98 Down CCCUGAGACCCUAACCUUAA miR-16- >hsa-miR-16-l-3p
SEQ ID MIMAT0004489
1 -3p No.19 0.1629 0,97 Down CCAGUAUUAACUGUGCUGCUGA miR- >hsa-miR-885-3p
SEQ ID MIMAT0004948
885-3p No.20 0.1744 1 ,04 Up AGGCAGCGGGGUGUAGUGGAUA miR- >hsa-miR-145-5p
SEQ ID MIMAT0000437
145-5p No.21 0, 1333 0,97 Down GUCCAGUUUUCCCAGGAAUCCCU miR- >hsa-miR-1247-5p
SEQ ID MIMAT0005899
1247- No.22 ACCCGUCCCGUUCGUCCCCGGA
0.0515 1 ,06 Up
5p miR- >hsa-miR-8089
SEQ ID MIMAT0031016
8089 No.23 0.4334 0.97 Down CCUGGGGACAGGGGAUUGGGGCAG miR- >hsa-miR-4292
SEQ ID MIMAT0016919
4292 No.24 0.3504 0,97 Down CCCCUGGGCCGGCCUUGG miR- >hsa-miR-6797-3p
SEQ ID MIMAT0027495
6797- No.25
UGCAUGACCCUUCCCUCCCCAC
0.0492 1.05 Up
3p miR- >hsa-miR-4646-5p
SEQ ID MIMAT0019707
4646- No.26 ACUGGGAAGAGGAGCUGAGGGA
0.3751 0.97 Down
5p miR- >hsa-miR-21 l-3p
SEQ ID MIMAT0022694
211 -3p No.27 0.2755 0.97 Down GCAGGGACAGCAAAGGGGUGC miR- >hsa-miR-3124-5p
SEQ ID MIMAT0014986
3124- No.28
UUCGCGGGCGAAGGCAAAGUC
0,7101 0.99 Down
5p miR- >hsa-miR-8078
SEQ ID MIMAT0031005
8078 No.29 0.0708 0.95 Down GGUCUAGGCCCGGUGAGAGACUC
For miRNA in miRBase there is the sequence of precursor: pre-miRNA (mir-xxx) and the two sequence of miRNA Mature: 5p and 3p (miR-xxx-3p and miR-xxx-5p) and the prefix for the species of origin: hsa-xxx for Homo sapiens.
For example: mir-204
The human pre-miRNA has the following sequence: hsa-mir-204 MI0000284
GGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUUUGUCAUCCUAUGCCUGAGAAUAUAUGAAGGAGGCUG GGAAGGCAAAGGGACGUUCAAUUGUCAUCACUGGC (SEQ ID. No.30)
The human mature 5p miRNA has the following sequence: hsa-miR-204-5p MIMAT0000265 uucccuuuGucAuccuAUGCCu (SEQ. ID No . l ) as listed in Table 1 .
The human mature 3p miRNA has the following sequence: hsa-miR-204-3p MIMAT0022693
GCUGGGAAGGCAAAGGGACGU (SEQ ID No. 31)
mir-501
The human pre-miRNA has the following sequence: hsa-mir-501 MI0003185
GCUCUUCCUCUCUAAUCCUUUGUCCCUGGGUGAGAGUGCUUUCUGAAUGCAAUGCACCCGGGCAAGGAUUCUGAG AGGGUGAGC (SEQ ID No.32)
The human mature 5p miRNA has the following sequence: hsa-miR-501 -5p MIMAT0002872 AAuccuuuGucccuGGGUGAGA (SEQ ID No .2 ) as listed in Table 1
The human mature 3p miRNA has the following sequence: hsa-miR-501 -3p MIMAT0004774 AAUGCACCCGGGCAAGGAUUCU (SEQ ID No.33) mir-136
The human pre-miRNA has the following sequence: hsa-mir-136 MI0000475
UGAGCCCUCGGAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUAUGCUCCAUCAUCGUCUCAAAUGAGUCUUCAGA GGGUUCU (SEQ ID No.34)
The human mature 5p miRNA has the following sequence:hsa-miR-136-5p MIMAT0000448
ACUCCAUUUGUUUUGAUGAUGGA (SEQ ID No.35)
The human mature 3p miRNA has the following sequence:hsa-miR-136-3p MIMAT0004606
CAucAucGucucAAAUGAGucu (SEQ ID No.3) as listed in Table 1. mir-34A
The human pre-miRNA has the following sequence: hsa-mir-34a MI0000268
GGCCAGCUGUGAGUGUUUCUUUGGCAGUGUCUUAGCUGGUUGUUGUGAGCAAUAGUAAGGAAGCAAUCAGCAAGU AUACUGCCCUAGAAGUGCUGCACGUUGUGGGGCCC (SEQ ID NO.36) .
The human mature 5p miRNA has the following sequence: hsa-miR-34a-5p MIMAT0000255 uGGCAGUGucuuAGCUGGuuGu (SEQ ID No.4) as listed in Table 1.
The human mature 3p miRNA has the following sequence: hsa-miR-34a-3p MIMAT0004557
CAAUCAGCAAGUAUACUGCCCU (SEQ ID No.37) . miRNA-204-5p There is no known function of this miRNA.
In one embodiment, the miRNA-204-5p is human.
In one embodiment, the miRNA-204-5p is measured in a blood sample, preferably a plasma sample.
In one embodiment, the miRNA-204-5p is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
An example RNA sequence of miRNA-204-5p is the hsa-miR-204-5p MIMAT0000265 UUCCCUUUGUCAUCCUAUGCCU (SEQ. ID o.l) . miRNA-501 -5p
There is no known function of this miRNA.
In one embodiment, the miRNA-501 -5p is human. In one embodiment, the miRNA-501 -5p is measured in a blood sample, preferably a plasma sample.
In one embodiment, the miRNA-501 -5p is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
An example RNA sequence of miRNA-501 -5p is hsa-miR-501 -5p MIMAT0002872 AAUCCUUUGUCCCUGGGUGAGA (SEQ ID No .2 ) . miRNA-136-3p
There is an association of this miRNA with Alzheimers and Parkinson's disease in US20140272993 but no mention of association with BBB impairment or risk of BBB impairment. In one embodiment, the miRNA-136-3p is human.
In one embodiment, the miRNA-136-3p is measured in a blood sample, preferably a plasma sample.
In one embodiment, the miRNA-136-3p is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment. An example RNA sequence of miRNA-136-3p is the hsa-miR-136-3p MIMAT0004606
CAUCAUCGUCUCAAAUGAGUCU (SEQ ID No .3 ) . miRNA-34-5p
There is an association of this miRNA with BBB function as disclosed in EP261 1918. In one embodiment, the miRNA-34-5p is human. In one embodiment, the miRNA-34-5p is measured in a blood sample, preferably a plasma sample.
In one embodiment, the miRNA-34-5p is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment. An example RNA sequence of miRNA-34-5p is hsa-miR-34a-5p MIMAT0000255
UGGCAGUGUCUUAGCUGGUUGU (SEQ ID No .4 ) .
Phosphatidylinositol-glycan-specific phospholipase D (PHLD)
Phosphatidylinositol-glycan-specific phospholipase D (PHLD) hydrolyzes the inositol phosphate linkage in proteins anchored by phosphatidylinositol glycans (GPI-anchor) thus releasing these proteins from the membrane.
In one embodiment, the PHLD is human PHLD.
In one embodiment, the PHLD is measured in a human blood sample, preferable a plasma sample.
An example amino acid sequence of PHLD is the sequence deposited under NCBI Accession No. NP_001494.2.
A further example of the amino acid sequence of PHLD is:
SEQ ID No.38
MSAFRLWPGLLIMLGSLCHRGSPCGLSTHVEIGHRALEFLQLHNGRVNYRELLLEHQDAYQAGIVFPDCFYPSIC KGGKFHDVSESTHWTPFLNASVHYIRENYPLPWEKDTEKLVAFLFGITSHMAADVSWHSLGLEQGFLRTMGAIDF HGSYSEAHSAGDFGGDVLSQFEFNFNYLARRWYVPVKDLLGIYEKLYGRKVITENVIVDCSHIQFLEMYGEMLAV SKLYPTYSTKSPFLVEQFQEYFLGGLDDMAFWSTNIYHL SFMLENG SDCNLPENPLFIACGGQQNHTQGSKMQ KNDFHRNLTTSLTESVDRNINYTERGVFFSVNSWTPDSMSFIYKALERNIRTMFIGGSQLSQKHVSSPLASYFLS FPYARLGWAMTSADLNQDGHGDLWGAPGYSRPGHIHIGRVYLIYGNDLGLPPVDLDLDKEAHRILEGFQPSGRF GSALAVLDFNVDGVPDLAVGAPSVGSEQLTYKGAVYVYFGSKQGGMSSSPNITISCQDIYCNLGWTLLAADVNGD SEPDLVIGSPFAPGGGKQKGIVAAFYSGPSLSDKEKLNVEAANWTVRGEEDFSWFGYSLHGVTVDNRTLLLVGSP TWKNASRLGHLLHIRDEKKSLGRVYGYFPPNGQSWFTISGDKAMGKLGTSLSSGHVLMNGTLKQVLLVGAPTYDD VSKVAFLTVTLHQGGATRMYAL SDAQPLLLSTFSGDRRFSRFGGVLHLSDLDDDGLDEI IMAAPLRIADV SGL IGGEDGRVYVYNGKETTLGDMTGKCKSWITPCPEEKAQYVLISPEASSRFGSSLITVRSKAKNQWIAAGRSSLG ARLSGALHVYSLGSD
PHLD may be processed into a mature form, for example by cleavage of a signal peptide. Thus, a further example amino acid sequence of PHLD is:
SEQ ID No.39
CGLSTHVEIGHRALEFLQLHNGRVNYRELLLEHQDAYQAGIVFPDCFYPSICKGGKFHDVSESTHWTPFLNASVH YIRENYPLPWEKDTEKLVAFLFGITSHMAADVSWHSLGLEQGFLRTMGAIDFHGSYSEAHSAGDFGGDVLSQFEF NFNYLARRWYVPVKDLLGIYEKLYGRKVITENVIVDCSHIQFLEMYGEMLAVSKLYPTYSTKSPFLVEQFQEYFL GGLDDMAFWSTNIYHLTSFMLENGTSDCNLPENPLFIACGGQQNHTQGSKMQ
KNDFHRNLTTSLTESVDRNINYTERGVFFSVNSWTPDSMSFIYKALERNIRTMFIGGSQLSQKHVSSPLASYFLS FPYARLGWAMTSADLNQDGHGDLWGAPGYSRPGHIHIGRVYLIYGNDLGLPPVDLDLDKEAHRILEGFQPSGRF GSALAVLDFNVDGVPDLAVGAPSVGSEQLTYKGAVYVYFGSKQGGMSSSPNITISCQDIYCNLGWTLLAADVNGD SEPDLVIGSPFAPGGGKQKGIVAAFYSGPSLSDKEKLNVEAANWTVRGEEDFSWFGYSLHGVTVDNRTLLLVGSP TWKNASRLGHLLHIRDEKKSLGRVYGYFPPNGQSWFTISGDKAMGKLGTSLSSGHVLMNGTLKQVLLVGAPTYDD VSKVAFLTVTLHQGGATRMYAL SDAQPLLLSTFSGDRRFSRFGGVLHLSDLDDDGLDEI IMAAPLRIADV SGL IGGEDGRVYVYNGKETTLGDMTGKCKSWITPCPEEKAQYVLISPEASSRFGSSLITVRSKAKNQWIAAGRSSLG ARLSGALHVYSLGSD
A further examples of amino acid sequence of PHLD isoform 2 is: SEQ ID No.40
MSAFRLWPGLLIMLGSLCHRGSPCGLSTHVEIGHRALEFLQLHNGRVNYRELLLEHQDAYQAGIVFPDCFYPSIC KGGKFHDVSESTHWTPFLNASVHYIRENYPLPWEKDTEKLVAFLFGITSHMAADVSWHSLGLEQGFLRTMGAIDF HGSYSEAHSAGDFGTVYLHLLNFLW
In one embodiment, the PHLD level is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment. Strontium (Sr)
Strontium (Sr) is an essential element with the largest proportion of strontium located in the bones (>99%). In biological fluids such as serum or plasma it can be bound to proteins.
In one embodiment, Strontium is measured in a human blood sample, preferably a serum sample. In one embodiment, the Strontium level is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
Proteoglycan 4 (PRG4)
Proteoglycan 4 plays a role in boundary lubrication within articulating joints. It prevents protein deposition onto cartilage from synovial fluid by controlling adhesion-dependent synovial growth and inhibiting the adhesion of synovial cells to the cartilage surface. Isoform F plays a role as a growth factor acting on the primitive cells of both hematopoietic and endothelial cell lineages.
In one embodiment, the Proteoglycan 4 (PRG4) is human PRG4.
In one embodiment, the PRG4 is measured in a blood sample, preferably a plasma sample. A number of isoforms are known: Isoform A, Isoform B, Isoform C, Isoform D, Isoform E, and Isoform F.
An example amino acid sequence of PRG4 is the sequence deposited under NCBI Accession No. NP_005798.3.
A further example of the amino acid sequence of PRG4 is: SEQ ID No.41
MAWKTLPIYLLLLLSVFVIQQVSSQDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPDFKRVCTAELSCKGRCF ESFERGRECDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSSKKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIE SEEITEEHSVSENQESSSSSSSSSSSSTIRKIKSSKNSAANRELQKKLKVKDNKKNRTKKKPTPKPPWDEAGSG LDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDG KEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPALTTPKEPTPTTPKEPASTTPKEPTPTTIKSAP TTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKE PAPTTPKEPTPTTPKEPAPTTKEPAPTTPKEPAPTAPKKPAPTTPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPT TTKSAPTTTKEPAPTTTKSAPTTPKEPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKP APTTPKEPAPTTPKETAPTTPKKLTPTTPEKLAPTTPEKPAPTTPEELAPTTPEEPTPTTPEEPAPTTPKAAAPN TPKEPAPTTPKEPAPTTPKEPAPTTPKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKEPTSTTCDKPA PTTPKGTAPTTPKEPAPTTPKEPAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKGPTSTTSDKPAPTTPKE TAPTTPKEPAPTTPKKPAPTTPETPPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPT TKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQVTSTTTQDTTPFKITT LKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTP TPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVNPKSEDAGGAEGETPHMLLRPHVFMPEVTPDMDYL PRVPNQGI I INPMLSDETNICNGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRI EVWGIPSPIDTVFT RCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQ EPVQKCPGRRPALNYPVYGETTQVRRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNW TSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSGQTLSKVWYNCP
PRG4 may be processed into a mature form, for example by cleavage of a signal peptide. Thus, a further example amino acid sequence of PRG4 is:
SEQ ID No.42
QDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPDFKRVCTAELSCKGRCFESFERGRECDCDAQCKKYDKCCPD YESFCAEVHNPTSPPSSKKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSS SSSTIRKIKSSKNSAANRELQKKLKVKDNKKNRTKKKPTPKPPWDEAGSGLDNGDFKVTTPDTSTTQHNKVSTS PKITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPT SKVLAKPTPKAETTTKGPALTTPKEPTPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPT TTKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPAPTTKEP APTTPKEPAPTAPKKPAPTTPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTKSAPTTP KEPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKPAPTTPKEPAPTTPKETAPTTPKKL TPTTPEKLAPTTPEKPAPTTPEELAPTTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPT TPKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKEPTSTTCDKPAPTTPKGTAPTTPKEPAPTTPKEPA PTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPAPTTPET PPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTERDL RTTPETTTAAPKMTKETATTTEKTTESKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMN KPEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQ TTTRPNQ PNSKLVEVNPKSEDAGGAEGE PHMLLRPHVFMPEV PDMDYLPRVPNQGI I INPMLSDETNICNGK PVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIK DAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGETTQV RRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNWTSAISLPNIRKPDGYDYYAFSKDQ YYNIDVPSRTARAITTRSGQTLSKVWYNCP
A further example of amino acid sequence of PRG4 isoform B is:
SEQ ID No.43
MAWKTLPIYLLLLLSVFVIQQVSSQELSCKGRCFESFERGRECDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSS KKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSSSSSTIRKIKSSKNSAAN RELQKKLKVKDNKKNRTKKKPTPKPPWDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPP NSDTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKG PALTTPKEPTPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTT TKEPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPAPTTKEPAPTTPKEPAPTAPKKPA PTTPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTKSAPTTPKEPSPTTTKEPAPTTPK EPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKPAPTTPKEPAPTTPKETAPTTPKKLTPTTPEKLAPTTPEKPA PTTPEELAPTTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPTTPKETAPTTPKGTAPTT LKEPAPTTPKKPAPKELAPTTTKEPTSTTCDKPAPTTPKGTAPTTPKEPAPTTPKEPAPTTPKGTAPTTLKEPAP TTPKKPAPKELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPAPTTPETPPPTTSEVSTPTTTKEP TTIHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKET ATTTEKTTESKI ATTTQV STTTQDT PFKI TLKTTTLAPKVTTTKK I TTEIMNKPEETAKPKDRATNSKA TTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVN PKSEDAGGAEGE PHMLLRPHVFMPEV PDMDYLPRVPNQGI I INPMLSDETNICNGKPVDGLTTLRNGTLVAFR GHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTG QIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGETTQVRRRRFERAIGPSQTHTI RIQYSPARLAYQDKGVLHNEVKVSILWRGLPNWTSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTR SGQTLSKVWYNCP
A further example of amino acid sequence of PRG4 isoform C is:
SEQ ID No.44
MAWKTLPIYLLLLLSVFVIQQVSSQDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPDFKRVCTAELSCKGRCF ESFERGRECDCDAQCKKYDKCCPDYESFCAEVKDNKKNRTKKKPTPKPPWDEAGSGLDNGDFKVTTPDTSTTQH NKVSTSPKITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSA KDLAPTSKVLAKPTPKAETTTKGPALTTPKEPTPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTP KEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPA PTTKEPAPTTPKEPAPTAPKKPAPTTPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTK SAPTTPKEPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKPAPTTPKEPAPTTPKETAP TTPKKLTPTTPEKLAPTTPEKPAPTTPEELAPTTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTP KEPAPTTPKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKEPTSTTCDKPAPTTPKGTAPTTPKEPAPT TPKEPAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPA PTTPETPPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDK TTERDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTIT TTEIMNKPEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRI AEAMLQTTTRPNQ PNSKLVEVNPKSEDAGGAEGE PHMLLRPHVFMPEV PDMDYLPRVPNQGI I INPMLSDET NICNGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWR FTNDIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVY GETTQVRRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNWTSAISLPNIRKPDGYDYY AFSKDQYYNIDVPSRTARAITTRSGQTLSKVWYNCP A further example of amino acid sequence of PRG4 isoform D is:
SEQ ID No.45
MAWKTLPIYLLLLLSVFVIQQVSSQELSCKGRCFESFERGRECDCDAQCKKYDKCCPDYESFCAEVKDNKKNRTK KKPTPKPPWDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPPNSDTSKETSLTVNKETTV ETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPALTTPKEPTPTTPKEPA STTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKE PAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPAPTTKEPAPTTPKEPAPTAPKKPAPTTPKEPAPTTPKEPAPT TTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTKSAPTTPKEPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEP APTTPKEPAPTTTKKPAPTTPKEPAPTTPKETAPTTPKKLTPTTPEKLAPTTPEKPAPTTPEELAPTTPEEPTPT TPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPTTPKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELA PTTTKEPTSTTCDKPAPTTPKGTAPTTPKEPAPTTPKEPAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKG PTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPAPTTPETPPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEP TPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQ VTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATTPKPQKPTKAPKKPTST KKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVNPKSEDAGGAEGETPHMLL RPHVFMPEV PDMDYLPRVPNQGI I INPMLSDETNICNGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARR ITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESV YFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGETTQVRRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLH NEVKVSILWRGLPNWTSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSGQTLSKVWYNCP
A further example of amino acid sequence of PRG4 isoform E is:
SEQ ID No.46
MAWKTLPIYLLLLLSVFVIQQVSSQELSCKGRCFESFERGRECDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSS KKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSSSSSTIRKIKSSKNSAAN RELQKKLKVKDNKKNRTKKKPTPKPPWDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPP NSDTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKG PALTTPKEPTPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTT PETPPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTE RDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTE IMNKPEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEA MLQTTTRPNQ PNSKLVEVNPKSEDAGGAEGE PHMLLRPHVFMPEV PDMDYLPRVPNQGI I INPMLSDETNIC NGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTN DIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGET TQVRRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNWTSAISLPNIRKPDGYDYYAFS KDQYYNIDVPSRTARAITTRSGQTLSKVWYNCP
A further example of amino acid sequence of PRG4 isoform F is:
SEQ ID No.47
MAWKTLPIYLLLLLSVFVIQQVSSQDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPDFKRVCTAELSCKGRCF ESFERGRECDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSSKKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIE SEEITEVKDNKKNRTKKKPTPKPPWDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPPNS DTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPA LTTPKEPTPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTK EPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPAPTTKEPAPTTPKEPAPTAPKKPAPT TPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTKSAPTTPKEPSPTTTKEPAPTTPKEP APTTPKKPAPTTPKEPAPTTPKEPAPTTTKKPAPTTPKEPAPTTPKETAPTTPKKLTPTTPEKLAPTTPEKPAPT TPEELAPTTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPTTPKETAPTTPKGTAPTTLK EPAPTTPKKPAPKELAPTTTKEPTSTTCDKPAPTTPKGTAPTTPKEPAPTTPKEPAPTTPKGTAPTTLKEPAPTT PKKPAPKELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPAPTTPETPPPTTSEVSTPTTTKEPTT IHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETAT TTEKTTESKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATT PKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVNPK SEDAGGAEGETPHMLLRPHVFMPEVTPDMDYLPRVPNQGI I INPMLSDETNICNGKPVDGL TLRNGTLVAFRGH YFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQI VAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGETTQVRRRRFERAIGPSQTHTIRI QYSPARLAYQDKGVLHNEVKVSILWRGLPNWTSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSG QTLSKVWYNCP
In one embodiment, Proteoglycan 4 level is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
Cholesterol ester 17:1
Cholesterol ester 17:1 is also known as 17:1 cholesteryl ester or CE(17:1 ). It is a cholesterol fatty acid ester or simply a cholesterol ester (CE). Cholesterol esters are cholesterol molecules with long-chain fatty acids linked to the hydroxyl group. They are much less polar than free cholesterol and appear to be the preferred form for transport in plasma and for storage. Cholesterol esters are major constituents of the adrenal glands and they also accumulate in the fatty lesions of atherosclerotic plaques. Cholesterol esters are also major constituents of the lipoprotein particles carried in blood (HDL, LDL, VLDL). The cholesterol esters in high- density lipoproteins (HDL) are synthesized largely by transfer of fatty acids to cholesterol from position sn-2 (or C-2) of phosphatidylcholine catalyzed by the enzyme lecithin cholesterol acyl transferase (LCAT). The enzyme also promotes the transfer of cholesterol from cells to HDL. As cholesterol esters accumulate in the lipoprotein core, cholesterol is removed from its surface thus promoting the flow of cholesterol from cell membranes into HDL. This in turn leads to morphological changes in HDL, which grow and become spherical. Subsequently, cholesterol esters are transferred to the other lipoprotein fractions LDL and VLDL, a reaction catalyzed by cholesteryl ester transfer protein.
An example of 17:1 cholesterol ester is PubChem CID: 24779603 with molecular formula C44H7602 and InChl Key RLMIGWIAENJHMP-RJRTUNKTSA-N.
In one embodiment, cholesterol ester 17:1 is measured in a blood sample, preferably plasma sample.
In one embodiment, cholesterol ester 17:1 is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment. Peroxiredoxin-2 (P32119 (PRDX2_HUMAN)) Peroxiredoxin-2 (PRDX2) is involved in redox regulation of the cell. It reduces peroxides with reducing equivalents provided through the thioredoxin system. It is not able to receive electrons from glutaredoxin and it plays an important role in eliminating peroxides generated during metabolism. It participates in the signalling cascades of growth factors and tumor necrosis factor-alpha by regulating the intracellular concentrations of peroxide.
In one embodiment, the Peroxiredoxin-2 (PRDX2) is human.
In one embodiment, the PRDX2 is measured in a blood sample, preferably a plasma sample.
An example amino acid sequence of PRDX2 is the sequence deposited under NCBI Accession No. NP_005800.3 A further example of the amino acid sequence of PRDX2 is:
SEQ ID No. 48
MASGNARIGKPAPDFKATAWDGAFKEVKLSDYKGKYWLFFYPLDFTFVCPTEI IAFSNRAEDFRKLGCEVLGV SVDSQFTHLAWIN PRKEGGLGPLNIPLLADVTRRLSEDYGVLKTDEGIAYRGLFI IDGKGVLRQI VNDLPVGR SVDEALRLVQAFQYTDEHGEVCPAGWKPGSDTIKPNVDDSKEYFSKHN
PRDX2 may be processed into a mature form, for example by cleavage of a signal peptide. Thus, a further example amino acid sequence of PRDX2 is:
SEQ ID N0.49
ASGNARIGKPAPDFKATAWDGAFKEVKLSDYKGKYWLFFYPLDFTFVCPTEI IAFSNRAEDFRKLGCEVLGVS VDSQFTHLAWIN PRKEGGLGPLNIPLLADVTRRLSEDYGVLKTDEGIAYRGLFI IDGKGVLRQI VNDLPVGRS VDEALRLVQAFQYTDEHGEVCPAGWKPGSDTIKPNVDDSKEYFSKHN
A further example of amino acid sequence of PRDX2 isoform 2 is: SEQ ID No.50
MASGNARIGKPAPDFKATAWDGAFKEVKLSDYKGKYWLFFYPLDFTFVCPTEI IAFSNRAEDFRKLGCEVLGV SVDSQFTHLAWYEQGPKREVAAKLTPSGPSSVASWPLLNLWNLRFPIVKIMETLPPKSLRMMTVISI
In one embodiment, PRDX2 level is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
Interleukin 16 (IL-16) lnterleukin-16 (IL-16) stimulates a migratory response in CD4+ lymphocytes, monocytes, and eosinophils. Primes CD4+ T-cells for IL-2 and IL-15 responsiveness. Also induces T- lymphocyte expression of interleukin 2 receptor. Ligand for CD4. Isoform 1 may act as a scaffolding protein that anchors ion channels in the membrane. Isoform 3 is involved in cell cycle progression in T-cells. Appears to be involved in transcriptional regulation of SKP2 and is probably part of a transcriptional repression complex on the core promoter of the SKP2 gene. May act as a scaffold for GABPB1 (the DNA-binding subunit the GABP transcription factor complex) and HDAC3 thus maintaining transcriptional repression and blocking cell cycle progression in resting T-cells. In one embodiment, the IL-16 is human IL-16.
In one embodiment, the IL16 is measured in a blood sample, preferably a serum sample.
An example amino acid sequence of IL-16 is the sequence deposited under NCBI Accession No. NP_757366.2.
A further example amino acid sequence of IL-16 is: SEQ ID No.51
MESHSRAGKSRKSAKFRSISRSLMLCNAKTSDDGSSPDEKYPDPFEISLAQGKEGIFHSSVQLADTSEAGPSSVP DLALASEAAQLQAAGNDRGKTCRRIFFMKESSTASSREKPGKLEAQSSNFLFPKACHQRARSNSTSVNPYCTREI DFPMTKKSAAPTDRQPYSLCSNRKSLSQQLDCPAGKAAGTSRPTRSLSTAQLVQPSGGLQASVISNIVLMKGQAK GLGFSIVGGKDSIYGPIGIYVKTIFAGGAAAADGRLQEGDEILELNGESMAGLTHQDALQKFKQAKKGLLTLTVR TRLTAPPSLCSHLSPPLCRSLSSSTCITKDSSSFALESPSAPISTAKPNYRIMVEVSLQKEAGVGLGIGLCSVPY FQCISGIFVHTLSPGSVAHLDGRLRCGDEIVEISDSPVHCLTLNEVY ILSHCDPGPVPI IVSRHPDPQVSEQQL KEAVAQAVENTKFGKERHQWSLEGVKRLESSWHGRPTLEKEREKNSAPPHRRAQKVMIRSSSDSSYMSGSPGGSP GSGSAEKPSSDVDISTHSPSLPLAREPWLSIASSRLPQESPPLPESRDSHPPLRLKKSFEILVRKPMSSKPKPP PRKYFKSDSDPQKSLEERENSSCSSGHTPPTCGQEARELLPLLLPQEDTAGRSPSASAGCPGPGIGPQTKSSTEG EPGWRRASPVTQTSPIKHPLLKRQARMDYSFDTTAEDPWVRISDCIKNLFSPIMSENHGHMPLQPNASLNEEEGT QGHPDGTPPKLDTANGTPKVYKSADSSTVKKGPPVAPKPAWFRQSLKGLRNRASDPRGLPDPALSTQPAPASREH LGSHIRASSSSSSIRQRISSFETFGSSQLPDKGAQRLSLQPSSGEAAKPLGKHEEGRFSGLLGRGAAPTLVPQQP EQVLSSGSPAASEARDPGVSESPPPGRQPNQKTLPPGPDPLLRLLSTQAEESQGPVLKMPSQRARSFPLTRSQSC ETKLLDEKTSKLYSISSQVSSAVMKSLLCLPSSISCAQTPCIPKEGASPTSSSNEDSAANGSAETSALDTGFSLN LSELREYTEGLTEAKEDDDGDHSSLQSGQSVISLLSSEELKKLIEEVKVLDEATLKQLDGIHVTILHKEEGAGLG FSLAGGADLENKVITVHRVFPNGLASQEGTIQKGNEVLSINGKSLKGTTHHDALAILRQAREPRQAVIVTRKLTP EAMPDLNSSTDSAASASAASDVSVESTAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASEQS ETVQPGDEILQLGGTAMQGLTRFEAWNI IKALPDGPV IVIRRKSLQSKETTAAGDS
IL-16 may be processed into a mature form, for example by cleavage of a signal peptide. Thus, a further example amino acid sequence of IL-16 is:
SEQ ID No.52
SAASASAASDVSVESTAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASEQSETVQPGDEILQ LGGTAMQGLTRFEAWNI IKALPDGPVTIVIRRKSLQSKETTAAGDS A further example of amino acid sequence of IL-16 isoform 2 is: SEQ ID No.53
MESHSRAGKSRKSAKFRSISRSLMLCNAKTSDDGSSPDEKYPDPFEISLAQGKEGIFHSSVQLADTSEAGPSSVP DLALASEAAQLQAAGNDRGKTCRRIFFMKESSTASSREKPGKLEAQSSNFLFPKACHQRARSNSTSVNPYCTREI DFPMTKKSAAPTDRQPYSLCSNRKSLSQQLDCPAGKAAGTSRPTRSLSTAQLVQPSGGLQASVISNIVLMKGQAK GLGFSIVGGKDSIYGPIGIYVKTIFAGGAAAADGRLQEGDEILELNGESMAGLTHQDALQKFKQAKKGLLTLTVR TRLTAPPSLCSHLSPPLCRSLSSSTCITKDSSSFALESPSAPISTAKPNYRIMVEVSLQKEAGVGLGIGLCSVPY FQCISGIFVHTLSPGSVAHLDGRLRCGDEIVEISDSPVHCLTLNEVY ILSHCDPGPVPI IVSRHPDPQVSEQQL KEAVAQAVENTKFGKERHQWSLEGVKRLESSWHGRPTLEKEREKNSAPPHRRAQKVMIRSSSDSSYMSGSPGGSP GSGSAEKPSSDVDISTHSPSLPLAREPWLSIASSRLPQESPPLPESRDSHPPLRLKKSFEILVRKPMSSKPKPP PRKYFKSDSDPQKSLEERENSSCSSGHTPPTCGQEARELLPLLLPQEDTAGRSPSASAGCPGPGIGPQTKSSTEG EPGWRRASPVTQTSPIKHPLLKRQARMDYSFDTTAEDPWVRISDCIKNLFSPIMSENHGHMPLQPNASLNEEEGT QGHPDGTPPKLDTANGTPKVYKSADSSTVKKGPPVAPKPAWFRQSLKGLRNRASDPRGLPDPALSTQPAPASREH LGSHIRASSSSSSIRQRISSFETFGSSQLPDKGAQRLSLQPSSGEAAKPLGKHEEGRFSGLLGRGAAPTLVPQQP EQVLSSGSPAASEARDPGVSESPPPGRQPNQKTLPPGPDPLLRLLSTQAEESQGPVLKMPSQRARSFPLTRSQSC ETKLLDEKTSKLYSISSQVSSAVMKSLLCLPSSISCAQTPCIPKEGASPTSSSNEDSAANGSAETSALDTGFSLN LSELREYTEGLTEAKEDDDGDHSSLQSGQSVISLLSSEELKKLIEEVKVLDEATLKQLDGIHVTILHKEEGAGLG FSLAGGADLENKVITVHRVFPNGLASQEGTIQKGNEVLSINGKSLKGTTHHDALAILRQAREPRQAVIVTRKLTP EAMPDLNSSTDSAASASAASDVSVESTEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASEQSE TVQPGDEILQLGGTAMQGLTRFEAWNI IKALPDGPV IVIRRKSLQSKETTAAGDS
A further example of amino acid sequence of IL-16 isoform 3 is:
SEQ ID No.54 MDYSFDTTAEDPWVRISDCIKNLFSPIMSENHGHMPLQPNASLNEEEGTQGHPDGTPPKLDTANGTPKVYKSADS STVKKGPPVAPKPAWFRQSLKGLRNRASDPRGLPDPALSTQPAPASREHLGSHIRASSSSSSIRQRISSFETFGS SQLPDKGAQRLSLQPSSGEAAKPLGKHEEGRFSGLLGRGAAPTLVPQQPEQVLSSGSPAASEARDPGVSESPPPG RQPNQKTLPPGPDPLLRLLSTQAEESQGPVLKMPSQRARSFPLTRSQSCETKLLDEKTSKLYSISSQVSSAVMKS LLCLPSSISCAQTPCIPKEGASPTSSSNEDSAANGSAETSALDTGFSLNLSELREYTEGLTEAKEDDDGDHSSLQ SGQSVISLLSSEELKKLIEEVKVLDEATLKQLDGIHVTILHKEEGAGLGFSLAGGADLENKVITVHRVFPNGLAS QEGTIQKGNEVLSINGKSLKGTTHHDALAILRQAREPRQAVIVTRKLTPEAMPDLNSSTDSAASASAASDVSVES TAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASEQSETVQPGDEILQLGGTAMQGLTRFEAW NI IKALPDGPVTIVIRRKSLQSKETTAAGDS A further example of amino acid sequence of IL-16 isoform 4 is: SEQ ID No.55
MDYSFDTTAEDPWVRISDCIKNLFSPIMSENHGHMPLQPNASLNEEEGTQGHPDGTPPKLDTANGTPKVYKSADS STVKKGPPVAPKPAWFRQSLKGLRNRASDPRGLPDPALSTQPAPASREHLGSHIRASSSSSSIRQRISSFETFGS SQLPDKGAQRLSLQPSSGEAAKPLGKHEEGRFSGLLGRGAAPTLVPQQPEQVLSSGSPAASEARDPGVSESPPPG RQPNQKTLPPGPDPLLRLLSTQAEESQGPVLKMPSQRARSFPLTRSQSCETKLLDEKTSKLYSISSQVSSAVMKS LLCLPSSISCAQTPCIPKEGASPTSSSNEDSAANGSAETSALDTGFSLNLSELREYTEGLTEAKEDDDGDHSSLQ SGQSVISLLSSEELKKLIEEVKVLDEATLKQLDGIHVTILHKEEGAGLGFSLAGGADLENKVITVHRVFPNGLAS QEGTIQKGNEVLSINGKSLKGTTHHDALAILRQAREPRQAVIVTRKLTPEAMPDLNSSTDSAASASAASDVSVES TEATVCTVTLEDVGRAGLQPGRREGLPTRRQASHH In one embodiment, IL-16 level is increased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
Tryptophan
Tryptophan (Trp) an essential amino acid in humans and animals, meaning the body cannot synthesize it and thus it must be obtained from the diet. Tryptophan is also a precursor to the neurotransmitters serotonin and melatonin.
In one embodiment, tryptophan is measured in a human blood sample, preferably plasma sample.
In one embodiment, tryptophan level is decreased relative to a reference value when the subject has an impaired BBB or increased risk of BBB impairment.
Determining biomarker levels
The level of the individual biomarker species in the sample may be measured or determined by any suitable method known in the art. For example, mass spectrometry (MS), antibody- based detection methods (e.g., enzyme-linked immunosorbent assay, ELISA), non-antibody protein scaffold-based methods (e.g., fibronectin scaffolds), radioimmunoassays (RIA) or aptamer-based methods may be used. Other spectroscopic methods, chromatographic methods, labelling techniques or quantitative chemical methods may also be used.
In one embodiment, the level of the one or more biomarkers may be determined via binding to one or more antibodies that are specific to the one or more biomarkers. Suitable antibodies are known or may be generated using known techniques.
Suitable methods for detecting antibody levels include, but are not limited to, immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, Western blotting and immunoprecipitation.
Preferably, the level of the one or more biomarkers is determined using a sandwich immunoassay.
The antibody may be, for example, a monoclonal antibody, polyclonal antibody, multispecific antibody (e.g., bispecific antibody) or fragment thereof provided that it specifically binds to the biomarker being detected. Antibodies may be obtained by standard techniques comprising immunising an animal with a target antigen and isolating the antibody from serum. Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. (Kohler et al. (1975) Nature 256: 495) or may be made by recombinant DNA methods (e.g. disclosed in US 4816567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (Clackson et al. (1991 ) Nature 352: 624-628) and Marks et al. (Marks et al. (1991 ) J. Mol. Biol. 222: 581 -597), for example. The antibody may also be a chimeric or humanised antibody. In one embodiment, the level of the one or more biomarkers may be determined by staining the sample with a reagent that labels one or more of the biomarkers. "Staining" is typically a histological method, which renders the biomarker detectable, for example by microscopic techniques, such as those using visible or fluorescent light.
In one embodiment, the biomarker is detected in the sample by immunohistochemistry (IHC). In IHC, the biomarker may be detected by an antibody that binds specifically to one or more of the biomarkers.
Two general methods of antibody-based detection (including for IHC-based methods) are available: direct and indirect assays. According to the first assay, binding of antibody to the target antigen is determined directly. This direct assay uses a labelled reagent, such as a fluorescent tag or an enzyme-labelled primary antibody, which can be visualised without further antibody interaction.
In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labelled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromogenic or fluorogenic substrate is added to provide visualisation of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
The primary and/or secondary antibody used may be labelled with a detectable moiety. Numerous labels are available, including radioisotopes, colloidal gold particles, fluorescent labels and various enzyme-substrate labels. Fluorescent labels include, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin and phycocyanin, and/or derivatives of any one or more of the above. The fluorescent labels can be conjugated to the antibody using known techniques.
Various enzyme-substrate labels are available (e.g., disclosed in US 4275149). The enzyme generally catalyses a chemical alteration of the chromogenic substrate that can be detected microscopically, for example under visible light. For example, the enzyme may catalyse a colour change in a substrate, or may alter the fluorescence or chemiluminescence of the substrate. Examples of enzymatic labels include luciferases (e.g. firefly luciferase and bacterial luciferase; e.g. disclosed in US 4737456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g. uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are well known.
Typically IHC methods may comprise a step of detecting stained regions within an image. Pixels in the image corresponding to staining associated with the biomarker may be identified by colour transformation methods, for example as disclosed in US 6553135 and US 6404916. In such methods, stained objects of interest may be identified by recognising the distinctive colour associated with the stain. The method may comprise transforming pixels of the image to a different colour space and applying a threshold value to suppress background staining. For example, a ratio of two of the RGB signal values may be formed to provide a means for discriminating colour information. A particular stain may be discriminated from background by the presence of a minimum value for a particular signal ratio. For example, pixels corresponding to a predominantly red stain may be identified by a ratio of red divided by blue (R/B) which is greater than a minimum value.
Kong et al. (Kong et al. (2013) Am. J. Clin. Nutr. 98: 1385-94) describes the use of the avidin- biotin-peroxidase method and two independent investigators counting the number of positively stained cells.
Detection using aptamers may comprise the following steps: aptamers that specifically recognise the biomarker may be synthesised using standard nucleic acid synthesis techniques or selected from a large random sequence pool, for example using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique; aptamers are mixed with the samples so that aptamer-protein complexes are formed; non-specific complexes are separated; bound aptamers are removed from their target proteins; aptamers are collected and measured, for example using microarrays or mass spectrometry techniques. Aptamers can be single stranded DNA or RNA sequences that fold into a unique 3D structure having a combination of stems, loops, quadruplexes, pseudoknots, bulges or hairpins. The molecular recognition of aptamers results from intermolecular interactions, such as the stacking of aromatic rings, electrostatic and van der Waals interactions, or hydrogen bonding with a target compound. In addition, the specific interaction between an aptamer and its target is complemented through an induced fit mechanism, which requires the aptamer to adopt a unique folded structure to its target. Aptamers can be modified to be linked with labelling molecules such as dyes or immobilised on the surface of beads or substrates for different applications. Samples
The invention comprises a step of determining the level of one or more biomarkers in one or more samples obtained from a subject.
Preferably, the sample is a sample derived from blood.
The sample derived from blood may contain a blood fraction or may be whole blood. Preferably, the sample derived from blood is a plasma or serum sample, most preferably a serum sample.
Techniques for collecting samples from a subject are well known in the art. Subject
The subjects disclosed herein are preferably mammals, particularly preferably humans. Both human and veterinary applications are within the scope of the invention.
In one embodiment, the subject may be at risk of developing an impaired blood-brain barrier secondary to any of the following conditions: traumatic brain injury, hypoxic ischemia, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
In another embodiment, the subject may be at risk of developing an impaired blood-brain barrier associated with vascular cognitive impairment, vascular dementia, age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
The subject may be, for example, an ageing human subject, such as a human over the age of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 years old. Preferably, the subject is a human over the age of 55 years old. For veterinary applications, the age of the animal would be scaled from the human situation using the average lifespan for calibration.
Method of treatment
It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment; although in the context of the invention references to preventing are more commonly associated with prophylactic treatment. Treatment may also include arresting progression in the severity of a disease.
Dietary intervention
The term "dietary intervention" refers to an external factor applied to a subject which causes a change in the subject's diet.
In one embodiment, the dietary intervention is a diet supplemented with vitamins and/or minerals, preferably vitamin B.
In another embodiment, the dietary intervention is a diet supplemented with omega-3 fatty acids. In one embodiment, the dietary intervention comprises increasing vitamin B intake by the subject, preferably by administering a vitamin B supplement.
In another embodiment, the dietary intervention comprises increasing omega-3 fatty acid intake by the subject, preferably by administering an omega-3 fatty acid supplement.
The vitamin B may be, for example, vitamin B12, vitamin B6 and/or folic acid. The omega-3 fatty acid may be, for example, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), preferably EPA.
The diet may be one which is adjusted to the starting body weight of the subject.
The dietary intervention may comprise administration of at least one diet product. The diet product may be a meal replacement product or a supplement product. The diet product may include food products, drinks, pet food products, food supplements, nutraceuticals, food additives or nutritional formulae.
EXAMPLES
Example 1 Materials and methods
Subject population
120 community-dwelling adults aged 55 years or older (48 of them having no cognitive impairment, 72 with cognitive impairment (mild cognitive impairment (MCI), n = 63; and mild dementia, n = 9) were enrolled in this study. The clinical evaluation included a neurological and general examination and extensive neuropsychological evaluation. Subjects with neurological or psychiatric diseases or with a severe or unstable medical illness were excluded. Along with the clinical examination, the Hospital Anxiety and Depression (HAD) scale was administered (Zigmond & Snaith, Acta Psychiatr. Scand. 1983, 67 (6), 361 -370). The study participants with MCI and the participants with mild dementia have been recruited among outpatients with cognitive impairment referred to the Memory Clinics, Departments of Psychiatry, and the Leenaards Memory Center, Department of Clinical Neurosciences, University Hospitals of Lausanne for investigation of their cognitive complaints. The diagnosis of MCI or of mild dementia was based on neuropsychological and clinical evaluation, and made by a consensus conference of psychiatrists and/or neurologists, and neuropsychologists prior to the inclusion in the study. For instance, MCI criteria required memory impairment (< 1.5 SD below the age, gender and education adjusted mean on the Buschke Double Memory Test verbal memory score) (Buschke, Sliwinski, Kuslansky, & Lipton, Neurology 1997, 48 (4), 989-997), and/or impairment in another cognitive domain such as executive tasks, and a Clinical Dementia Rating (CDR) (Morris, Neurology 1993, 43 (1 1 ), 2412-2414) equal to 0.5.
Probable Alzheimer's dementia was defined according to the clinical diagnostic criteria from the National Institute on Aging and Alzheimer's Association and DSM-IV criteria for dementia of the Alzheimer type (American-Psychiatric-Association). Participants in this group have a CDR of 1.0. The participants without cognitive impairment (n = 48) had no history or evidence of cognitive decline, and a CDR score of 0. They are community-dwelling volunteers recruited by advertisement or among the spouses of memory clinic patients.
Neuropsychological and functional assessments
The neuropsychological assessment includes measures of memory and other major cognitive domains such as language, attention and executive functioning. This assessment consists of the Mini Mental State Examination (Folstein MF et al. 1975, J. Psychiatr. Res 12, 189-198), the Buschke Double Memory Test (Buschke H et al. 1997, Neurology, 48, 989- 997) the digit span forward and backward (Wisdom NM et al. 2012, Arch Clin
Neuropsychology 27, 389-397), the Stroop Test (Stroop JR 1935, J. of Expt. Psychology 18, 643-662), the letter fluency task (Cardebat D et al. 1990, Acta Neurol Belg 90, 207-217), and the Trail Making Tests A and B (Reitan RM 1955, J. Consult Psychol 19, 393-394). The functional assessment includes the ADL and instrumental ADL (IADL) (Lawton MP et al. 1969, Gerontologist 9, 179-186), as well as the CDR (Morris JC 1993, Neurology, 43, 2412- 2414). The neuropsychological test battery, ADL and IADL, and the CDR were used to verify inclusion and exclusion criteria.
Additional assessment
The brief clinical form of the Neuropsychiatric Inventory (Kaufer, D.I. et al. (2000) J. Neuropsychiatry Clin. Neurosci. 12: 233-9) was administrated to assess neuropsychiatric symptoms in all participants. The Cumulative illness rating scale-geriatrics (Miller MD et al. 1992, Psychiatry Res 41 ,237-248) was used to measure the participant individual chronic medical illness burden.
Cerebrospinal fluid (CSF) and blood collection and handling
Venous and lumbar punctures were performed between 8:30 and 9:30 am in the Memory centres after an overnight fast. Blood was drawn into EDTA containing vacutainers (Sarstedt, Germany) and spun down to permit aliquots of supernatant (plasma and serum) for the analysis. Lumbar puncture and spinal fluid collection was performed on subjects in sitting or lying position with a 22G "atraumatical" spinal needle to capture 10-12 mL of CSF into polypropylene tubes. CSF cell count and protein quantification were performed in 2-3 mL and the remaining CSF was centrifuged, aliquoted, snap-frozen and stored at -80 °C until assay.
Neuroinflammatory biomarker analysis - IL-16
A "sandwich" immunoassay (Meso Scale Discovery (MSD), Rockville, MD, USA) quantified 37 analytes (IFN-gamma, IL-1 B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TNFa, IL-1 a, IL-5, IL-7, IL- 12/23p40, IL-15, IL-16, IL-17A, TNF-B, VEGFA, Eotaxin, MIP-1 B, Eotaxin-3, TARC, IP-10, MIP-1 a, MCP-1 , MDC, MCP-4, VEGF-C,VEGF-D, Tie-2, Flt-1 , PIGF, bFGF, SAA, CRP, VCAM-1 , ICAM-1 ) in serum.
Samples were measured following the manufacturer's instructions. Briefly, the 96-well plates pre-coated with capture antibodies were blocked with 5% MSD Blocker A Solution. Calibrator dilutions were prepared and samples were diluted as recommended for each kit with MSD Diluents. Samples and calibrators were then added to the plates and incubated at room temperature with shaking for 2 h. Plates were washed three times with a home-prepared solution of 10 x phosphate-buffered saline (PBS), pH 7.4 (Corning, Manassas, VA, USA)- Tween 20 (Fisher Scientific, Pittsburgh, PA, USA). Detection antibodies were mixed with MSD Diluents as indicated in the protocols of each kit and incubated at room temperature with shaking for 1 -2 h. Plates were washed three times with the PBS-Tween 20 solution. MSD Read buffer was added and plates were read on an MSD instrument (SECTOR Imager 6000 reader). Data were generated and interpolated using MSD Discovery Workbench software.
Proteomic Biomarker Analysis
Human plasma samples were analysed according to a recently published protocol (Dayon, L. et al. (2014) J. Proteome Res.. 13: 3837-45).
From 25 μΙ_ of plasma sample (diluted in 75 μΙ_ of Buffer A (Agilent Technologies, Wilmington, DE, USA) containing 0.0134 mg-mL"1 LACB and filtered with 0.22 μηι filter plate from Millipore), 14 abundant plasma proteins were removed, following the manufacturer instructions, with MARS columns (Agilent Technologies) and high performance liquid chromatography (HPLC) systems (Thermo Scientific, San Jose, CA, USA) equipped with an HTC-PAL (CTC Analytics AG, Zwingen, Switzerland) fraction collectors. After immuno-depletion, samples were snap-freezed. Buffer exchange was performed with Strata-X 33u Polymeric reversed-phase (RP) (30mg/1 ml_) cartridges mounted on a 96-hole holder and a vacuum manifold, all from Phenomenex (Torrance, CA, USA). Samples were subsequently evaporated with a vacuum centrifuge (Thermo Scientific) and stored at -80 °C. Reduction with TCEP, alkylation with IAA, digestion with Lys-C/trypsin Promega (Promega, Madison, Wl, USA), TMT 6-plex (Thermo Scientific, Rockford, IL, USA) labelling, sample pooling, and SPE purification (Oasis HLB (Milford, MA, USA) and SCX (Phenomenex)) were performed on a 4-channels Microlab Star liquid handler (Hamilton, Bonaduz, Switzerland). The pooled 6-plex TMT-labelled samples were then evaporated to dryness before storage at -80 °C. The samples were dissolved in 500 μΙ_ H20/CH3CN/formic acid 96.9/3/0.1 for RP liquid chromatography tandem mass spectrometry (LC MS/MS). RP-LC MS/MS was performed with hybrid linear ion trap-Orbitrap (LTQ-OT) Elite and an Ultimate 3000 RSLC nano system (Thermo Scientific). Proteolytic peptides (injection of 5 μΙ_ of sample) were trapped on an Acclaim PepMap 75 μηη χ 2 cm (C18, 3 μηη, 100 A) pre-column and separated on an Acclaim PepMap RSLC 75 μηη χ 50 cm (C18, 2 μηη, 100 A) column (Thermo Scientific) coupled to a stainless steel nanobore emitter (40 mm, OD 1/32") mounted on a Nanospray Flex Ion Source
(Thermo Scientific). The analytical separation was run for 150 min using a gradient that reached 30% of CH3CN after 140 min and 80% of CH3CN after 150 min at a flow rate of 220 nL-min"1. For MS survey scans, the OT resolution was 120000 (ion population of 1 χ 106) with an m/z window from 300 to 1500. For MS/MS with higher-energy collisional dissociation (HCD) at 35% of the normalized collision energy, ion population was set to 1 χ 105 (isolation width of 2), with a resolution of 15000, first mass at m/z = 100, and a maximum injection time of 250 ms in the OT. A maximum of 10 precursor ions (most intense) were selected for MS/MS. Dynamic exclusion was set for 60 seconds within a ± 5 ppm window. A lock mass of m/z = 445.1200 was used. Each sample was analysed in duplicate.
Proteome Discoverer (version 1 .4, Thermo Scientific) was used as data analysis interface. Identification was performed against the human UniProtKB/Swiss-Prot database (08/12/2014 release) including the LACB sequence (20194 sequences in total). Mascot (version 2.4.2, Matrix Science, London, UK) was used. Variable amino acid modifications were oxidized methionine, deamidated asparagine/glutamine, and 6-plex TMT-labelled peptide amino terminus (+ 229.163 Da). 6-plex TMT-labelled lysine (+ 229.163 Da) was set as fixed modifications as well as carbamidomethylation of cysteine. Trypsin was selected as the proteolytic enzyme, with a maximum of two potential missed cleavages. Peptide and fragment ion tolerance were set to, respectively, 10 ppm and 0.02 Da. All Mascot result files were loaded into Scaffold Q+S 4.4.1.1 (Proteome Software, Portland, OR, USA) to be further searched with X! Tandem (version CYCLONE (2010.12.01.1 )). Both peptide and protein FDRs were fixed at 1 % maximum, with a 2 unique peptide criterion to report protein identification. Quantitative values were exported from Scaffold Q+S as log2 of the protein ratio fold changes with respect to their measurements in the biological reference, i.e., mean log2 values after isotopic purity correction but without normalization applied between samples and experiments. The biological reference was a pool of all individual plasma samples labelled with 6-plex TMT reporter-ions at m/z = 126 and 131 , allowing ratio fold change calculation with respect to both channels. Because of the concordance between both calculation results, the average of the values was done as well as the average of both replicate measurements.
Mineral Biomarker Analysis
Human serum samples were analysed according to a recently published protocol (Konz, T. et al. (2017) J. Proteome Res.. 16: 2080-90).
All ICP-MS experiments carried out in this study were performed using an Agilent 8800 triple quadrupole ICP-MS (Agilent Technologies, Tokyo, Japan) operated in low matrix plasma mode. The mass spectrometric device was equipped with an integrated autosampler, a concentric nebulizer and a Scott double-pass spray chamber. The instrument was tuned prior analysis by using a multi-element tuning solution to verify the functional conditions of the mass spectrometer. An internal standard (ISTD) solution containing beryllium (200 ng-mL"1), scandium, gallium, indium, tellurium and bismuth at 100 ng-mL"1 was mixed online with the samples via a T-connector.
All solutions were prepared by using 18 ΜΩ-crrr1 deionized water obtained from a Milli-Q system (Millipore, Bedford, MA, USA). All chemical substances used for analysis were of highest grade of purity available. The diluent solution was composed of 5% 1 -butanol (99.9%, Sigma-Aldrich, St. Louis, MO, USA), 0.05% EDTA (ethylenediaminetetraacetic acid, 99.995% trace metals basis, Sigma-Aldrich), 0.05% triton X-100 (BioXtra, Sigma-Aldrich) and 0.25% ammonium hydroxide (Sigma-Aldrich). Solutions for external calibration and online-internal standard were prepared from ICP standards (Merck Millipore, Darmstadt, Germany), while the tuning solution was purchased from Agilent Technologies (Santa Clara, CA, USA)
All sample preparation steps were conducted at room temperature. Before analysis, each serum sample was thawed only once and homogenized for 10 s using a vortex mixer. Subsequently, the samples were diluted (1 :10) using the diluent solution. For the preparation of the spiked serum samples, a human serum pool was aliquoted and spiked with ICP standards. Three different concentration levels, adjusted to the concentration range of the respective elements in human serum were prepared. To avoid cross contamination of the analytes, two different sets of spiked samples were prepared: set 1 consisting of level 1-3 (Mg, P, S, K, Ca and Mo) and set 2, level 1-3 (B, Al, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Cd, Sn, I, Cs, Ba Hg and Pb).
miRNA Biomarker Analysis
Human plasma samples were analysed following the manufacturer's instructions: HTG Molecular Diagnostics, Inc. (Tucson, AZ) for miR profiling using qNPA. The expression of 2,083 human miRs transcripts was analysed using HTG EdgeSeq miRNA Whole Transcriptome Assay and next-generation sequencing. The process consist of just five simples steps with low hands-on time.
Plasma sample (25 μΙ) are combined with 25μΙ of pre-warmed HTG Plasma Lysis Buffer and 1/10 Proteinase K and incubated for 3h at 50°c with orbital shaking. After sample preparation, they are processed using the automated HTG EdgeSeq system. Samples was hybridized with miRNA probes in qNPA reaction in which an excess of nuclease protection probes (NPPs) complimentary to each miRNA hybridize to their target. Briefly, following lysis of plasma samples, probes specific for the whole miRNA were incubated with the samples, forming specific probe-miRNA duplexes, then unhybridized probes were digested by S1 Nuclease, followed by alkaline hydrolysis to destroy the sample miRNA in the duplexes. This left intact probes with stoichiometric concentrations proportional to the abundance of specific miRNA in the original sample.
After samples removed from the HTG EdgeSeq system, the second step of library preparation is the PCR amplification of capture probes with barcoded sequencing adapter. Hemo KlenTaq® enzyme (NEB, M0332S) was used for plasma/serum sample. The index adapter are provided by supplier: HTG EdgeSeq sequencing tag Pack. The final library was purified with AMPure XP beads (Beckman Coulter, Brea, CA) in ratio x2.5. After quantification with Qubit system (Life Technologies) and profiling with LabChip DNA NGS 3K Assay (PerkinElmer, Inc), libraries were pooled equimolar at 2nM and by 24 samples for sequencing.
Sequencing was performed on a MiSeq lllumina platform (lllumina, San Diego, CA) with MiSeq® Reagent Kit v3 (150 cycle). EdgeSeq system generate sample sheet according with the run assay and specific for the chosen sequencer. Library pool are loaded at 20 pM final on standard MiSeq flowcell with 5% of PhiX spike and sequencing was performed for 1 x 50 cycles on a Miseq (lllumina) with 150V3 chemistry.
After sequencing, use the HTG EdgeSeq parser for post-sequencing data processing. Load the individual FastQ files generated by the sequencer to the parser. Probe sequences are aligned to the sequencing reads, making data processing simple and very quick. The system generate an excel sheet with the count for each miRNA probes tested (2,083) and for positive and negative control. The miRNA assay contains both positive and negative control probes. These probes are used to assess the performance of the chemistry to ensure quality data is produced. Raw count dataset were transfer to Quartz Bio S.A. for analysis.
Amino acid Biomarker Analysis
Sample preparation was done with 50 μΙ_ of blood plasma or serum with a mixture of 10 μΙ_ of labelled internal standards and 140 μΙ_ of ice-cold methanol (0.1 % formic acid) for protein precipitation were added. Samples were agitated by vortex (5 min) followed by centrifugation at 14500 rpm for 10 min at 4°C. The supernatant was then collected and subjected to derivatization.
Derivatization was performed using the AccQ-Tag Ultra Derivatization Kit Amino Acid Analysis (Waters Corp.) following manufacturer's instructions: 10 μΙ_ of either a standard amino acid mix solution or the supernatant of the sample was mixed with 70 μΙ_ of AccQ-Tag Ultra borate buffer (pH 8.8). The derivatization was carried out by adding 20 μΙ_ of reconstituted AccQ-Tag Ultra reagent (3 mg mL-1 of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, or AQC in acetonitrile) to the buffered mixture. The sample was then immediately vortexed followed by incubation at 55°C for 10 min.
Finally, the analysis of amino acids was performed using Ultra Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). UPLC-MS/MS analysis was performed on a Waters Acquity UPLC system coupled on-line to a Waters Xevo TQ mass spectrometer using an electrospray ionization (ESI) probe. Chromatographic separation was achieved using a Waters AccQ-Tag Ultra column (100 x 2.1 mm x, 1 .7 μηη particle size) using a binary system of eluents for gradient elution. Eluent A contained 10% of AccQ-Tag Ultra Eluent A concentrate (Waters Corp.) while eluent B was composed of 100% AccQ-Tag Ultra Eluent B (Waters Corp.). For gradient elution, the following conditions were applied: 0.5 min, 0% mobile phase B; 6 min, 9% mobile phase B; 7.8 min 20% mobile phase B; 8 min, 90% mobile phase B; 8.7 min, 0% mobile phase B. The autosampler temperature was set at 20 °C and the column temperature at 55°C. The sample injection volume was 2 μΙ_. The transition m/z=171 , corresponding to the common main product ion from the collision- induced dissociation of all the AQC adducts, was used for the quantification of individual amino acids. Retention times of amino acids including that of tryptophan were determined using injection of standard amino acid solutions into the UPLC-ESI-MS/MS system.
The UPLC-MS/MS system control and data acquisition were performed with the Waters Corporation MassLynxTM software. Data analysis was conducted with the TargetLynxTM software (Waters Corporation).
Lipid Biomarker Analysis
Human serum samples were analysed according to a recently published protocol (Surma, M.A. et al. (2015) Eur. J. Lipid Sci. TechnoL 1 17: 1540-49).
Lipid extraction was carried out in high grade polypropylene deep well plates. Fifty microliters of diluted plasma (50x) (equivalent of 1 mL of undiluted plasma) was mixed with 130 mL of ammonium bicarbonate solution and 810 mL of methyl tert-butyl ether/methanol (7:2, v/v) solution was added. Twenty-one microliters of internal standard mixture was pre-mixed with the organic solvents mixture. The internal standard mixture contained: 50 pmol of lysophasphatidylglycerol (LPG) 17:1 ,50 pmol of lysophosphatic acid (LPA) 17:0, 500 pmol of phosphatidylcholine (PC) 17:0/17:0, 30 pmol of hexosylcer-amide (HexCer) 18:1 ;2/12:0, 50 pmol of phosphatidylserine(PS) 17:0/17:0, 50 pmol of phosphatidylglycerol (PG) 17:0/17:0, 50 pmol of phosphatic acid (PA) 17:0/17:0, 50 pmol of lysophposphatidylinositol (LPI 17:1 ), 50 pmol of lysophos-phatidylserine (LPS) 17:1 , 1 nmol of cholesterol (Choi) D6,100 pmol of diacylglycerol (DAG) 17:0/17:0, 50 pmol of triacylglycerol (TAG) 17:0/17:0/17:0, 50 pmol of ceramide(Cer) 18:1 ;2/17:0, 200 pmol of sphingomyelin (SM) 18:1 ;2/12:0, 50 pmol of lysophosphatidylcholine (LPC) 12:0,30 pmol of lysophosphatidylethanolamine (LPE) 17:1 ,50 pmol of phosphatidylethanolamine (PE) 17:0/17:0,100 pmol of cholesterol ester (CE) 20:0, 50 pmol of phosphatidylinositol (PI) 16:0/16:0. The plate was then sealed with a teflon-coated lid, shaken at 4°C for 15 min, and spun down (3000 g, 5 min) to facilitate separation of the liquid phases and clean-up of the upper organic phase. Hundred microliters of the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. Dried lipids were re-suspended in 40 mL of 7.5 mM ammonium acetate in chloroform/methanol/propanol (1 :2:4, v/v/v) and the wells were sealed with an aluminum foil to avoid evaporation and contamination during infusion. All liquid handling steps were performed using Hamilton STARIet robotic platform with the AntiDroplet.
Samples were analysed by direct infusion in a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences, Ithaca, NY, USA). Five microliters were infused with gas pressure and voltage set to 1 .25 psi and 0.95 kV, respectively. The delivery time was set to 4 min and 55 s with contact closure delay of 20 s to avoid initial spray instability. Polarity switch from positive to negative mode was set at 135 s after contact closure. Samples were analysed in both polarities in a single acquisition. The MS acquisition method starts with positive ion mode by acquiring the m/z 402-412 in MS positive mode at resolution of 140000 to monitor the [Choi + NH4 +]+ ion for 12 s. All individual scans in every segment are the average of 2 micro-scans. Automatic gain control (AGC) was set to 5 x 105 and maximum ion injection time was set to 200 ms. Then we scan the m/z 550-1000 in MS positive mode (resolution of 140000) with lock mass activated at a common background (m/z = 680.48022) for 18 s. AGC was set to 106 and maximum ion injection time was set to 50 ms. This is followed by a MS/MS (resolution of 17500) data independent analysis triggered by an inclusion list for 105 s. The inclusion list contains all the masses from 500.5 to 999.75 with 1 Da intervals. AGC was set to 105 and maximum ion injection time was set to 64 ms. The isolation width was set to 1.0 Da, first mass of MS/MS acquisition was 250 Da and normalized collision energy was set to 20%. Both MS and MS/MS data are combined to monitor SE, DAG, and TAG ions as ammonium adducts. After polarity switch to negative ion mode, a lag of 15 s before acquisition was inserted to allow spray stabilization. Then, we scan for the m/z 400- 650 (resolution of 140000) for 15 s with lock mass activated at a common background (m/z = 529.46262) to monitor LPG, LPA, LPI, LPS, and LPE as deprotonated anions and LPC and LPC O- as acetate adducts. AGC was set to 106 and maximum ion injection time was set to 50 ms. We then scan the m/z 520-940 (resolution of 140000) for 15 s with lock mass activated at a common background (m/z = 529.46262). AGC was set to 106 and maximum ion injection time was set to 50 ms. Finally, we scan MS/MS (resolution of 17500) by data independent analysis triggered by an inclusion list for 90 s. This inclusion list contains all the masses from 590.5 to 939.5 with 1 Da intervals. AGC was set to 105 and maximum ion injection time was set to 64 ms. Isolation width was set to 1 .0 Da, first mass of MS/MS acquisition was 150 Da, and normalized collision energy was set to 35%. Both MS and MS/MS data were combined in order to monitor PC, PC 0-, HexCer, Cer, SM as acetate adducts and PS, PG, PA, PE, PE 0-, and PI as deprotonated anions.
All data were analysed with an in-house developed lipid identification software based on LipidXplorer (Herzod, R. et al. (2012) PloS One 7: e29851 ). Tolerance for MS and MS/MS identification was set to 2 ppm in scans where we have lock mass activated nd 8 ppm when lock mass was not available. Data post-processing and normalization were performed using an in-house developed data management system.
APOE genotyping
DNA was extracted from whole blood using the QIAsymphony DSP DNA Kit (Qiagen, Hombrechtikon, Switzerland). The SNV rs429358 and rs7412 were genotyped using the Taqman assays C_3084793_20 and C_904973_10 respectively (Thermo Fischer Scientific, Waltham, MA USA).
Ethical approvals for human research
The study was approved by the CHUV (Centre Hospitalier Universitaire Vaudois) Lausanne hospital ethics committee and the Canton of Vaud, Switzerland, Commission Cantonale d'ethique de la recherche sur I'etre humain (CER-VD). Written informed consent was obtained from all study participants.
Statistical analysis - Pre-analytical quality control of biomarker data
Biomarker data was quality-controlled prior to hypothesis testing by first excluding those with more than 5% missing data. The remaining missing data (< 5%) was imputed by randomly drawing a measure between the observed range of biomarker values. Biomarker data was then log-transformed to approach a Gaussian distribution, and standardised prior to final hypothesis testing.
Statistical analysis - Reference model of BBB impairment
The association of BBB impairment with demographic variables age, gender, education, ApoE4 presence, CSF Abeta, CSF p-tau, CSF tau, CDR category, and status in terms of diabetes, hypertension, and hypercholesterolemia was analysed using logistic regression models. The performance of the obtained classifier was assessed by measuring (i) its area under the Receiver Operating Characteristic (ROC) curve and its 95% confidence interval (using a bootstrap approach with 1000 iterations) and (ii) its accuracy (cumulated proportion of true-positives and true-negatives in the obtained 2x2 confusion matrix). The accuracy of this model was 86.4% with an AUC of 0.75.
BBB impairment was defined as CSF-to-serum ratio of albumin greater than 9.0.
Statistical analysis - Best model multi-omics of BBB impairment
The best predictive model takes into account the 4 miRNA and other 7 biomarkers. The miRNA biomarkers were miR-204-5p, miR-501 -5p, miR-136-3p, miR-34a-5p, together with other blood-based biomarkers phosphatidylinositol-glycan-specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , peroxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp) have been determined as biomarkers for identifying BBB impairment or risk of developing BBB impairment. The accuracy of this model was 93.0% with an AUC if 0.96. Least absolute shrinkage and selection operator (LASSO) logistic regression selected biomarkers that best predict BBB impairment. A reference model was initially generated, testing variables that are likely to be available to the clinicians to provide a benchmark for comparison with the models that included blood biomarkers. A 10-fold cross- validation process was performed for each LASSO analysis, which allows estimating the confidence interval of the misclassification error for each value of the regularization parameter λ. The LASSO analyses were repeated 100 times (1000 times for the reference model). The models that minimized the upper limit of the cross-validated misclassification error confidence interval across the 100 runs with less than 20 features were selected. Their performance was assessed by receiver operating characteristic (ROC) area under the curve (AUC) estimation using a bootstrap approach with 1000 iterations. Results were compared visually and formally tested for significance against the reference model using ROC AUC and accuracy using a McNemar test.
Results and discussion
Baseline characteristics are shown in Table 2. 1 18 subjects passed pre-analytical quality control for missing data, of which 13.5% (n = 16) met the criteria for blood-brain barrier (BBB) impairment. There were no significant differences between age, education, MMSE, HAD scale, CDR, presence of ApoEe4 allele, CSF abetai-42, CSF t-tau and CSF phospho-tau181 between subjects with and without BBB impairment. However, there were more men with BBB impairment. Consistent with the literature, subjects with CDR 0.5/1 compared to CDR 0 had significantly higher albumin ratio verifying the functional significance of BBB function. Table 2. Clinical and demographic characteristics of the older adult po pulation1
Figure imgf000052_0001
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.

Claims

1 . A method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least one miRNA in the biological sample from the subject c) comparing the level of expression of at least one miRNA in the sample to a level of expression of the miRNA in a reference wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
2. The method according to claim 1 wherein the at least one miRNA is selected from the group of miRNAs comprising Table 1 .
3. The method according to claim 1 or claim 2 wherein the at least one miRNA is selected from the group consisting of: miRNA 204-5p, miRNA 501 -5p, miR136-3p or miRNA 34a-5p.
4. The method according to any one of claims 1 to 3 wherein the at least one miRNA is selected from miRNA204-5p or miRNA501 -5p.
5. A method of identifying a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) comprising: a) isolating a biological sample from the subject b) measuring the level of expression of at least two miRNAs in the biological sample from the subject c) comparing the level of expression of at least two miRNAs in the sample to a level of expression of the miRNA in a reference wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB).
6. The method according to claim 5 wherein the at least two miRNA are selected from the group of miRNAs comprising Table 1 .
7. The method according to claim 6 wherein the at least two miRNAs are selected from the group consisting of: miRNA 204-5p, miRNA 501 -5p, miRNA136-3p or miRNA 34a-5p.
8. The method according to any one of claims 6 to 7 wherein the at least two miRNAs are selected from miRNA204-5p and miRNA501 -5p.
9. The method according to any preceding claim 1 -8 wherein the method further comprises determining the level at least one further biomarker selected from the group consisting of: phosphatidylinositol-glycan specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , perioxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp) in one or more samples obtained from the subject.
10. The method according to any preceding claim 1 - 9 wherein the method further comprises determining a blood-brain barrier (BBB) impairment score (S) using the formula:
S = A x (miR-204-5p) + B x (miR-501 -5p) + C x (miR-136-3p) + D x (miR-34a-5p) + E x (PHLD) + F x (strontium) + G (PRG4) + H (cholestrol ester 17:1 ) + I x (PRDX2) + J x (IL16) + K x (Trp) wherein A, B, C, D, E, F, and G, H, I, J and K are coefficients.
1 1 . The method according to any preceding claim 1 -10 wherein the reference is a sample from a normal healthy subject without blood brain barrier impairment.
12. The method according to any one of claims 1 -1 1 wherein the biological sample is selected from the group consisting of a bodily fluid, tissue or cell.
13. The method according to any one of claims 1 -12 wherein said bodily fluid sample is a blood sample.
14. The method according to any one of claims 1-13 wherein said bodily fluid is a serum or plasma sample.
15. The method according to any one of claims 1 -14 wherein the identification of a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) is associated with vascular cognitive impairment, vascular dementia, age-related cognitive decline, Alzheimer's disease (AD) and Parkinson's disease (PD).
16. The method according to any one of claims 1 -14 wherein the identification of a subject with an impaired blood-brain barrier (BBB) or at risk of developing an impaired blood-brain barrier (BBB) is associated with any of the following conditions: traumatic brain injury, hypoxic ischemia, septic encephalopathy, brain tumours, systemic inflammation, diabetes mellitus, hypertension, cerebral ischemia, acute kidney injury, viral infection, parasitic infection, pharmaceutical and environmental exposure to chemicals and nutritional deficiencies.
17. A method of treating or preventing blood brain barrier (BBB) impairment comprising the steps: a) determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB) according to the method of any one of claims 1 to 16; and b) applying an intervention capable of improving blood-brain barrier (BBB) function to a subject identified to be in need thereof.
18. The method of claim 17 wherein the intervention is a dietary intervention.
19. The method of claim 18 wherein the dietary intervention comprises increasing vitamin B intake by the subject, preferably by administering a vitamin B supplement.
20. The method of claim 18-19 wherein the dietary intervention comprises increasing omega- 3 fatty acid intake by the subject, preferably by administering an omega-3 fatty acid supplement.
21. A kit of parts for determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB), wherein the kit comprises one or more biomarkers selected from the group consisting of: miRNA 204-5p, miRNA 501 - 5p, miRNA136-3p, miRNA 34a-5p, phosphatidylinositol-glycan specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , perioxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp).
22. A kit of parts for determining whether a subject has an impaired blood-brain barrier (BBB) or is at risk of developing an impaired blood-brain barrier (BBB), wherein the kit comprises two or more biomarkers selected from the group consisting of: miRNA 204-5p, miRNA 501 -5p, miRNA136-3p, miRNA 34a-5p, phosphatidylinositol-glycan specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , perioxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp).
23. A kit of parts for determining whether a subject is at risk of developing a cognitive impairment, wherein the kit comprises one or more biomarkers selected from the group consisting of: miRNA 204-5p, miRNA 501 -5p, miRNA136-3p, miRNA 34a-5p, phosphatidylinositol-glycan specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , perioxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp).
24. A kit of parts for determining whether a subject is at risk of developing a cognitive impairment, wherein the kit comprises two or more biomarkers selected from the group consisting of: miRNA 204-5p, miRNA 501 -5p, miRNA136-3p, miRNA 34a-5p, phosphatidylinositol-glycan specific phospholipase D (PHLD), strontium (Sr), proteoglycan 4 (PRG4), cholesterol ester 17:1 , perioxiredoxin-2 (PRDX2), interleukin 16 (IL-16), and tryptophan (Trp).
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