Patent Application
20210270847
The invention relates generally to protein and higher molecular weight protein breakdown products (ranging from about 85% or less of the size of the intact proteins to greater than 10 kDa) and lower molecular weight peptide fragment (ranging from 500 Da to 10, kDa) biomarkers that are released into biological fluids and can be measured in fluid biological samples, such as cerebrospinal fluid, blood, dialysate, or central nervous system tissue lysate, after traumatic injury to the central nervous system. Specifically, particular discrete anatomical regions of the brain, cell types, subcellular structures, and brain extracellular matrix can be identified as damaged through detection of these markers. The invention therefore also encompasses methods of diagnosis, prognosis and management of central nervous system injury.
Injury to the central nervous system (CNS) occurs in a variety of medical conditions and in trauma, and has been the subject of intense scientific scrutiny in recent years. The brain has such high metabolic requirements that it can suffer permanent neurological damage if deprived of sufficient oxygen (hypoxia) for even a few minutes. Under conditions of hypoxia or anoxia, when mitochondrial production of ATP cannot meet the metabolic requirements of the brain, tissue damage occurs.
This process is exacerbated by neuronal release of the neurotransmitter glutamate, which stimulates NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and kainate receptors. Activation of these receptors initiates calcium influx into the neurons and production of reactive oxygen species, which are potent toxins that damage important cellular structures such as membranes, DNA and enzymes.
The brain has many redundant blood supplies, which means that its tissue is seldom completely deprived of oxygen, even during acute ischemic events caused by thromboembolic events or trauma. A combination of the injury of hypoxia with the added insult of glutamate toxicity therefore is believed to be ultimately responsible for cellular death, therefore, if glutamate toxicity can be alleviated, neurological damage could also be lessened. Antioxidants and anti-inflammatory agents have been proposed to reduce damage, but they often have poor access to structures such as the brain, which is protected by the blood brain barrier.
Brain injury, such as cerebral apoplexy, is a result of a sudden circulatory disorder of a human brain area with subsequent functional losses and corresponding neurological and/or psychological symptoms. Cerebral apoplexy can be caused by cerebral hemorrhages (e.g., after a vascular tear in hypertension, arteriosclerosis and apoplectic aneurysms) and ischemia (e.g., due to a blood pressure drop crisis or embolism), leading to degeneration or destruction of the brain cells. After a cerebral vascular occlusion, only part of the tissue volume is destroyed as a direct result of the restricted circulation and the associated decreased oxygen supply. The tissue area designated as the infarct core can only be kept from dying off by immediate re-canalization of the vascular closure, e.g., by local thrombolysis, and is therefore only accessible to therapy in a very limited fashion. The outer peripheral zone, referred to as the penumbra, loses its function immediately after onset of the vascular occlusion, but initially remains adequately supplied with oxygen by the collateral supply and becomes irreversibly damaged after only a few hours or days. Since the cell death in this area does not occur immediately, methods to block the damage after stroke and trauma have been investigated. However, without early diagnosis, the prognosis for such subjects is poor.
The mammalian nervous system comprises the peripheral nervous system (PNS) and the central nervous system (CNS, comprising the brain and spinal cord), and is composed of two principal classes of cells: neurons and glial cells. The glial cells fill the spaces between neurons, nourishing them and modulating their function. Certain glial cells, such as Schwann cells in the PNS and oligodendrocytes in the CNS, also provide a protective myelin sheath that surrounds and protects neuronal axons, the processes that extend from the neuron cell body and through which the electric impulses of the neuron are transported. In the peripheral nervous system, the long axons of multiple neurons are bundled together to form a nerve or nerve fiber. These in turn may be combined into fascicles, such that the nerve fibers form bundles embedded together with the intraneural vascular supply in a loose collagenous matrix bounded by a protective multilamellar sheath. In the central nervous system, the neuron cell bodies are visually distinguishable from their myelin-sheath processes, giving rise to the terms gray matter, referring to the neuron cell bodies, and white matter, referring to the myelin-covered processes.
During development, differentiating neurons from the central and peripheral nervous systems send out axons that must grow and make contact with specific target cells. In some cases, growing axons must cover enormous distances; some extend into the periphery, whereas others stay confined within the central nervous system. In mammals, this stage of neurogenesis is complete during the embryonic phase of life and neuronal cells do not multiply once they have fully differentiated. Accordingly, the neural pathways of a mammal are particularly at risk if neurons are subjected to mechanical or chemical trauma or neuropathic degeneration sufficient to put the neurons that define the pathway at risk of dying.
A host of neuropathies, some of which affect only a subpopulation or a system of neurons in the peripheral or central nervous systems, have been identified to date. The neuropathies, which may affect the neurons themselves or the associated glial cells, may result from cellular metabolic dysfunction, infection, exposure to toxic agents, autoimmunity dysfunction, malnutrition or ischemia. In some cases the cellular dysfunction is thought to induce cell death directly. In other cases, the neuropathy may induce sufficient tissue necrosis to stimulate the body's immune/inflammatory system and the body's immune response to the initial neural injury then destroys the neurons and the pathway defined by these neurons.
Another common injury to the CNS is stroke, the destruction of brain tissue as a result of intracerebral hemorrhage or infarction. Stroke is a leading cause of death in the developed world. Injury after stroke can be caused by reduced blood flow (ischemia or ischemic stroke) that results in deficient blood supply and death of tissues in one area of the brain (infarction). Causes of ischemic strokes include blood clots that form in the blood vessels in the brain (thrombi) and blood clots or pieces of atherosclerotic plaque or other material that travel to the brain from another location (emboli). Bleeding (hemorrhage) within the brain may also cause symptoms that mimic ischemic stroke.
Mammalian neural pathways also are at risk due to damage caused by neoplastic lesions. Neoplasias of both the neurons and glial cells have been identified. Transformed cells of neural origin generally lose their ability to behave as normal differentiated cells and can destroy neural pathways by loss of function. In addition, the proliferating tumors may induce lesions by distorting normal nerve tissue structure, inhibiting pathways by compressing nerves, inhibiting cerebrospinal fluid or blood supply flow, and/or by stimulating the body's immune response. Metastatic tumors, which are a significant cause of neoplastic lesions in the brain and spinal cord, may similarly damage neural pathways and induce neuronal cell death.
In 2010, about 2.5 million emergency department visits, hospitalizations or deaths were associated with traumatic brain injury (TBI), either alone or in combination with other injuries, in the United States. TBI contributed to the deaths of more than 50,000 people and was diagnosed in more than 280,000 hospitalizations.
Over the past decade (2001-2010), while rates of TBI-related emergency visits increased by 70%, hospitalization rates increased by only 11% and death rates decreased by 7%. In 2009, an estimated 248,418 children ages 20 or younger were treated for TBI in the United States. Emergency room visits for sports and recreation-related injuries included a diagnosis of concussion or TBI. From 2001 to 2009 the rate of emergency room visits for sports and recreation-related injuries with a diagnosis of concussion or TBI, alone or in combination with other injuries, rose 57% among children and young adults.
Chronic Traumatic Encephalopathy (CTE) is a progressive degenerative disease resulting from repetitive TBI. This type of injury was previously called punch-drunk syndrome or dementia pugilistica. CTE is commonly found in professional athletes participating in contact sports such as boxing, rugby, American football, ice hockey, and professional wrestling. It has also been found in soldiers exposed to blast or concussive injury. Symptoms associated with CTE may include dementia such as memory loss, aggression, confusion and depression, which generally appear years or decades after the trauma.
It has been hypothesized that the pathological process that leads to acute traumatic injury to the CNS consists of two steps. The primary injury results from the physical and mechanical force or blast overpressure wave as a result of direct impact to the CNS tissue. The secondary injury is the cascade of biochemical events such as proteolysis of cytoskeletal, membrane, and myelin proteins due to the elevation in intracellular Ca2+ that activates cysteine proteases such as calpain. The proteolysis causes progressive tissue degeneration, including neuronal cell death, axonal degeneration, and demyelination.
Neurological examinations are currently used for diagnosis, determination of severity, and prediction of neurological outcome in the brain injuries such as TBI and stroke. Although these tests can diagnose acute brain injury, assessment of injury severity and prognosis is often challenging. Current methods often cannot accurately assess the severity of TBI or predict long-term outcomes of TBI subjects. It also has been difficult to pinpoint the exact area of the brain or the cell type that has been injured. In addition, the neurological and functional recovery of TBI subjects is highly variable.
Therefore, there is a need in the art, not only for improved methods to diagnose traumatic injury to tissues of the central and peripheral nervous system, but also for new methods that can more discretely identify the nature and the extent of the injury for purposes of diagnosis and prognosis, and to guide treatment protocols.
Diagnostic clinical assessments of nervous system injury severity and therapeutic treatment efficacy have been studied, including biomarkers that can indicate brain damage and traumatic brain injury. The discovery and use of biomarkers for TBI is expected to lead to development of new therapeutic interventions that can be applied to prevent or reduce disability due to TBI. Biomarkers generated after brain damage have not been associated with specific regions or cell types, however. Identification of neurochemical markers specific to or predominantly found in the nervous system (CNS and PNS) would prove immensely beneficial for both prediction of outcome and guidance of targeted therapeutic delivery.
Therefore, the invention relates to a method of diagnosing trauma to the central nervous system in a subject in need thereof, comprising testing a first fluid biological sample obtained from the subject for the level of at least two proteins, protein breakdown products, or peptide fragments of one or more proteins selected from the group consisting of (a) Synapsin (Synapsin I, Synapsin II, Synapsin III); (b) Glutamate decarboxylase (GAD1; GAD2); (c) Golli-Myelin Basic Protein 1; (d) Golli-Myelin Basic Protein 1 in combination with classic Myelin Basic Protein Isoform 5; (e) Microtubule associated protein 6 (MAP6); (f) Neurogranin; (g) Vimentin; (h) Vimentin in combination with Glial Fibrillary Acidic Protein; (i) Tau-758 isoform; (j) Tau-758 isoform in combination with Tau-441 isoform; (k) Glial fibrillary acidic protein (GFAP); (1) Cortexin (Cortexin 1, Cortexin 2, Cortexin 3); (m) Striatin; (n) Neurexin (Neurexin-1, Neurexin-2, Neurexin-3); (o) Brain acidic soluble protein 1 (BASP1); (p) GAP43; (q) Calmodulin Regulated Spectrin Associated Protein (CAMSAP1, CAMSAP2, CAMSAP3); (r) Chondroitin Sulfate Proteoglycan 4; (s) Neurocan; and (t) Brevican; wherein levels of the at least two proteins or protein breakdown products that are at least two-fold higher in the fluid biological sample from the subject than the levels of the at least two proteins or protein breakdown products in a fluid biological sample from an uninjured subject indicate the presence of a central nervous system injury. In addition, the invention relates to a method of diagnosing trauma to the central nervous system in a subject in need thereof, comprising testing a first fluid biological sample obtained from the subject for the level of at least two proteins, protein breakdown products, or peptide fragments of one or more proteins selected from the group consisting of (a) Synapsin (Synapsin I, Synapsin II, Synapsin III); (b) Tau-441 isoform; (c) Tau-758 isoform; (d) Neurogranin; (e) Vimentin; (f) Classic Myelin Basic Protein Isoform 5; (g) Golli-Myelin Basic Protein 1; (h) Glial Fibrillary Acidic Protein; and (i) MAP6, (j) complement protein Clq (Clqa, Clqb, Clqc), C3, C5, C1s, C1QRF and complement receptor CR1; wherein levels of the at least two peptide fragments that are at least two-fold higher in the fluid biological sample from the subject than the levels of the at least two peptide fragments in a fluid biological sample from an uninjured subject indicate the presence of a central nervous system injury.
In preferred embodiments, the at least two peptide fragments are selected from the group consisting of:
AEPRQEFEVMEDHAGTYGLG;
SPQLATLADEVSASLAK;
ILDIPLDDPGANAAAAKIQAS(p)FRGHMARKKIKSGERGRKGPGPGGPG
GA;
ILDIPLDDPGANAAAAKIQASFRGHMARKKIKSGERGRKGPGPGGPGGA;
HGSKYLATASTMD;
NAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDE;
Also, in preferred embodiments, the first fluid biological sample is obtained from the subject within 24 hours of the trauma to the central nervous system or within 3 days of the trauma to the central nervous system. In other embodiments, the one or more additional fluid biological samples are obtained from the subject at subsequent times to the first fluid biological sample.
Preferably, the testing comprises subjecting the fluid biological samples are subjected to ultrafiltration using a ultrafiltration membrane filter with a molecular weight cutoff of about 10,000 Da to separate an ultrafiltrate fraction and then subjecting the ultrafiltrate fraction to assay for proteins, protein breakdown products or peptide fragments. In certain embodiments, an increasing level of the at least two proteins, protein breakdown products, or peptide fragments in fluid biological samples taken at subsequent times indicates worsening of the severity of the central nervous system injury; a decreasing level of the at least two proteins, protein breakdown products, or peptide fragments in fluid biological samples taken at subsequent times indicates improvement in the central nervous system injury; and an unchanging level of the at least two proteins, protein breakdown products, or peptide fragments in fluid biological samples taken at subsequent times indicates a leveling of the severity of the central nervous system injury.
Embodiments of the invention also include a method of identifying the anatomical location of trauma to the central nervous system in a subject in need thereof, comprising testing a fluid biological sample obtained from the subject for the presence of any combination of (a) one or more cortexin proteins, protein breakdown products, or peptide fragments, the presence of which above control levels identifies the cortex as the anatomical location; (b) one or more myelin basic protein proteins, protein breakdown products, or peptide fragments, the presence of which above control levels identifies the white matter as the anatomical location; and (c) one or more striatin proteins, protein breakdown products, or peptide fragments, the presence of which above control levels identifies the striatum as the anatomical location.
Further embodiments of the invention include a method of identifying cell types injured in trauma to the central nervous system in a subject in need thereof, comprising testing a fluid biological sample obtained from the subject for the presence of any combination of (a) one or more protein, or protein breakdown product of brain acidic soluble protein−1, glutamate decarboxylase 1, glutamate decarboxylase 2, neurochondrin or any combination thereof, the presence of which above control levels identifies the cell type as neurons; (b) one or more protein, or protein breakdown product of Vimentin, the presence of which above control levels identifies the cell type as astroglia; and (c) one or more protein, or protein breakdown product of myelin basic protein 5 or Golli-myelin basic protein, the presence of which above control levels identifies the cell type as oligodendrocytes and complent protein Clq (Clqa, Clqb, Clqc), C3, C5, C1s, Clq ligand and complment receptor CR1 from microglia cells. Additional embodiments include a method of identifying the subcellular location of injury to the central nervous system after trauma in a subject in need thereof, comprising testing a fluid biological sample obtained from the subject for the presence of any combination of (a) one or more protein, or protein breakdown product of neurexin-1, neurexin-2, neurexin-3, synapsin-I, synapsin-II, synapsin-III or any combination thereof, the presence of which above control levels identifies the subcellular location as the presynaptic terminal; (b) one or more protein, or protein breakdown product of neurogranin, the presence of which above control levels identifies the subcellular location as the post-synaptic terminal; (c) one or more protein, or protein breakdown product of brain acidic soluble protein 2, growth associated protein 43 or a combination thereof, the presence of which above control levels identifies the subcellular location as the growth cone; (d) one or more protein, or protein breakdown product of nesprin-1, the presence of which above control levels identifies the subcellular location as the neuronal nucleus; (e) one or more protein, or protein breakdown product of Calmodulin regulated spectrin-associated protein 1, Calmodulin regulated spectrin-associated protein 2, Calmodulin regulated spectrin-associated protein 3, or any combination thereof, the presence of which above control levels identifies the subcellular location as the cortical cytoskeleton and axon; (f) one or more protein, or protein breakdown product of microtubule associated protein 6, the presence of which above control levels identifies the subcellular location as dendrites; and (g) one or more protein, or protein breakdown product of chondroitin sulfate proteoglycan 4, neurocan, brevican or any combination thereof, the presence of which above control levels identifies the subcellular location as the extracellular matrix.
The invention also includes embodiments such as a method of diagnosing the severity of trauma to the central nervous system in a subject in need thereof, comprising the steps of (a) testing a first fluid biological sample obtained from the subject up to 3 days after central nervous system injury for the levels of one or more proteins, protein breakdown products, and peptide fragments derived from a protein selected from one or more of Synapsin I, Synapsin II, Synapsin III, Tau-441 isoform, Tau-758 isoform, neurogranin, Vimentin, myelin basic protein Isoform 5, Golli-myelin basic protein 1, complement protein Clq (Clqa, Clqb, Clqc), C3, C5, Cls, Clq ligand and complment receptor CR1 and glial fibrillary acidic protein; (b) testing a second subsequent fluid biological sample obtained from the subject subsequent to the first fluid biological sample for the levels of the same one or more proteins, protein breakdown products, and peptide fragments as step (a); (c) optionally testing further subsequent fluid biological samples for the levels of the same one or more proteins, protein breakdown products, and peptide fragments as step (a); (d) comparing the levels of the one or more proteins, protein breakdown products, and peptide fragments in the fluid biological samples to a control sample from an uninjured subject and to each other; and (e) when the levels of peptide breakdown products in the fluid biological samples increase in subsequent samples, diagnosing a severe central nervous system injury.
Embodiments of the invention include a method of distinguishing severe trauma to the central nervous system with pathoanatomical lesions detectable by CT, MRI, or both, from less severe central nervous system trauma with no detectable pathoanatomical lesions in a subject in need thereof, comprising (a) testing at least one first fluid biological sample obtained from the subject within 24 hours after central nervous system injury for the levels of one or more peptide fragments of a protein selected from one or more of Synapsin I, Synapsin II, Synapsin III, Tau-441 isoform, Tau-758 isoform, neurogranin, Vimentin, myelin basic protein isoform 5, Golli-myelin basic protein 1, a complement protein and glial fibrillary acidic protein; (b) testing a second subsequent fluid biological sample obtained from the subject about 2 days to about 6 months subsequent to the first fluid biological sample for the levels of the same one or more peptide fragments as step (a); (c) comparing the levels of the same one or more peptide fragments in the first and second fluid biological samples to a control sample from an uninjured subject and to each other; and (d) when the levels of the same one or more peptide fragments in the first fluid biological sample are above those in the control sample but decrease in the second fluid biological samples, diagnosing an acute central nervous system injury; and when the levels of the same one or more peptide fragments in the first fluid biological samples are above those in the control sample and increase or remain constant in subsequent samples, diagnosing a chronic central nervous system injury.
Embodiments of the invention also include a method of determining the damaged central nervous system anatomical areas, cell types and subcellular structures in a subject with central nervous system injury in need thereof, comprising (a) testing a fluid biological sample obtained from the subject after central nervous system injury for the levels of one or more proteins, protein breakdown products and/or peptide fragments of (1) a protein selected from cortexin-1, cortexin-2, cortexin-3 and any combination thereof; (2) a protein selected from myelin basic protein 5, Golli-myelin basic protein and a combination thereof; and (3) the protein striatin; (b) testing the fluid biological sample for the levels of one or more proteins, protein breakdown products and/or peptide fragments of (1) a protein selected from brain acidic soluble protein 1, glutamine decarboxylase 1, glutamate decarboxylase 2, neurochondrin or any combination thereof; (2) Vimentin; and (3) a protein selected from myelin basic protein 5, Golli-myelin basic protein and a combination thereof; and (c) testing the fluid biological sample for the levels of one or more proteins, protein breakdown products and/or peptide fragments of (1) a protein selected from cortexin-1, cortexin-2, cortexin-3, neurexin-1, neurexin-2, neurexin-3 and any combination thereof; (2) neurogranin; (3) BASP2/GAP43; (4) nesprin-1; (5) a protein selected from calmodulin regulated spectrin-associated protein 1, calmodulin regulated spectrin-associated protein 2, calmodulin regulated spectrin-associated protein 3, Tau-441, Tau-758 and any combination thereof; (6) microtubule associated protein 6; and (7) a protein selected from chondroitin sulfate proteoglycan 4, neurocan, brevican, or any combination thereof; wherein the presence of levels above control of cortexin-1, cortexin-2, or cortexin-3 is associated with cerebrocortical injury; the presence of levels above control of myelin basic protein 5 or Golli-myelin basic protein is associated with white matter or myelin sheath injury; the presence of levels above control of striatin is associated with striatum injury; the presence of levels above control of brain acidic soluble protein 1, glutamine decarboxylasel, glutamine decarboxylase 2 or neurochondrin is associated with neuronal cell body injury; the presence of levels above control of Vimentin is associated with astroglial injury; the presence of levels above control of myelin basic protein 5 or Golli-myelin basic protein is associated with oligodendrocyte injury; the presence of levels above control of cortexin-1, cortexin-2, cortexin-3, neurexin-1, neurexin-2, or neurexin-3 is associated with presynaptic terminal damage; the presence of levels above control of neurogranin is associated with post-synaptic terminal damage; the presence of levels above control of BASP2/GAP43 is associated with growth cone damage; the presence of levels above control of Nesprin-1 is associated with neuronal nuclear damage; the presence of levels above control of calmodulin regulated spectrin-associated protein 1, calmodulin regulated spectrin-associated protein 2, calmodulin regulated spectrin-associated protein 3, Tau-441, or Tau-758 is associated with axonal injury; the presence of levels above control of microtubule associated protein 6 is associated with dendritic damage; and the presence of levels above control of chondroitin sulfate proteoglycan 4, neurocan or brevican is associated with brain extracellular matrix damage; to determine the damaged central nervous system anatomical areas, cell types and subcellular structures in a subject associated with the one or more proteins, protein breakdown products and/or peptide fragments of present above control levels in the fluid biological sample.
Preferred embodiments of the invention are those wherein the trauma is cortical impact, closed head injury, blast overpressure induced brain injury, or concussion, and wherein the fluid biological sample is cerebrospinal fluid, blood, plasma, serum, wound fluid, or biopsy, necropsy or autopsy samples of brain tissue, spinal tissue, retinal tissue, and/or nerves.
Embodiments of the invention include a diagnostic kit comprising (a) detection agents for antibody, aptamer or mass spectrometry detection methods for detection of one or more peptide fragments selected from the group consisting of
AEPRQEFEVMEDHAGTYGLG;
SPQLATLADEVSASLAK;
ILDIPLDDPGANAAAAKIQAS(p)8FRGHMARKKIKSGERGRKGPGPGGP
GGA (*(p)=phospho-Serine);
ILDIPLDDPGANAAAAKIQASFRGHMARKKIKSGERGRKGPGPGGPGGA;
HGSKYLATASTMD;
NAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDE;
(b) an analyte protein, protein breakdown product, or peptide fragment to serve as internal standard and/or positive control; and (c) a signal generation coupling component.
The following figures are included to further demonstrate certain non-limiting embodiments of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
digestion. The sequences in the order shown are
Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood in the art to which this invention pertains and at the time of its filing. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. However, the skilled should understand that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, it should also be understood that as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration and any other measurements, quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary. Hence, where appropriate to the invention and as understood by those of skill in the art, it is proper to describe the various aspects of the invention using approximate or relative terms and terms of degree commonly employed in patent applications, such as: so dimensioned, about, approximately, substantially, essentially, consisting essentially of, comprising, and effective amount. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
As used herein, the terms “protein breakdown product” or “PBP” refer to a high molecular weight product of protein proteolysis, produced by one or more cleavages of a peptide bonds in the amino acid sequence, i.e., a product of protein cleavage, including chains of any length shorter than the native full-length sequence and longer than about 10,100 Da. The terms “peptide fragment,” or “PF” refer to a low molecular weight products of protein proteolysis, produced by one or more cleavages of a peptide bonds in the amino acid sequence, i.e., a product of protein cleavage. In one example, PFs may include fragments of the intact protein having 85 percent or less the size of the intact protein and greater than 10,000 Da. In another embodiment, PFs may include smaller fragments, i.e. including chains of any length shorter than about 10,000 Da, or 10,100 Da, or such peptide fragments that are able to pass through an ultrafiltration membrane with an approximate 10,000 Da cutoff, including PFs in the range of about 1,000 Da to about 10,000 Da, preferably about 2,000 to 8,000 Da, and most preferably about 2,000 to 5,000 Da. In general, a peptide fragment (PF), as used in this application, refers to an amino acid chain small enough to pass through an ultrafiltration membrane with an approximate 10,000 Da cutoff. As used herein, the term “analyte” and all of its cognates refers to any and all of the proteins, PBPs, or PFs that are analyzed or detected according to this invention.
The PFs and PBPs of the invention are referenced in this application by sequence, amino acid residue number from a protein, or by name. The invention, however, is intended to include peptides that are variants of these particular disclosed sequences. For example, minor differences such as deletion of one or two C- or N-terminal amino acids (or both) of the sequence are contemplated for use with the invention as peptide variants. Other minor differences such a an addition of one or two C- or N-terminal amino acids (or both) of the sequence likewise are contemplated for use with the invention. Minor differences which are caused by variable sequences of the protein, also are contemplated as part of the invention, including differences caused by natural differences in the protein sequence among species or among individuals are intended to be included in certain embodiments of the invention, as well.
As used herein, the phrase “trauma to the central nervous system,” “CNS trauma,” or “traumatic brain injury” includes any sudden injury to the brain, retina, spinal cord, or any part thereof, and includes injury to the projections (e.g., axons, dendrites, neurites) and subcellular parts of cells of the central nervous system due to trauma such as a physical impact or force, or a blast overpressure wave. Examples of CNS trauma include traumatic brain injury (TBI) or traumatic spinal cord injury (SCI). Much of the time, the injury will be the direct result of a traumatic injury, however the invention contemplates uses for injury or destruction of central nervous system tissue and/or cells indirectly caused by trauma, including but not limited to inflammation induced by trauma, swelling induced by trauma, or degenerative disease induced by trauma (such as CTE, Alzheimer's disease, Parkinsonianism, and the like).
As used herein, the term “subject in need” or “subject in need thereof” refers to any animal or a human subject that has been subjected to or suffers from a central nervous system trauma, or is suspected of suffering from a central nervous system injury as a result of trauma.
As used herein, the term “fluid biological sample” refers to a liquid or liquified sample obtained from a subject in need, and includes cerebrospinal fluid, whole blood, plasma, serum, wound fluid, and biopsy or autopsy samples of brain tissue, spinal tissue, retinal tissue, and/or nerves, such as tissue lysates. The samples preferably are prepared for analysis by, for example, centrifugation and/or filtration, preferably by ultrafiltration.
As used herein, in the term “testing a fluid biological sample of the subject for the level” and the term “levels” in the context of test results, “level” refers to the amount or concentration of a target analyte such as a peptide in a fluid biological sample.
As used herein, in the term “anatomical location” refers to a major central nervous system area, such as cortex, hippocampus, striatum, corpus callosum, cerebellum, retina, spinal cord, and the like, but also to cell type such as neuron, glia, astrocyte and the like, and to subcellular regions such as axon, dendrite, extracellular matrix, neuronal nucleus, cortical cytoskeleton and the like.
It is to be understood that in instances where a range of values are provided that the range is intended to encompass not only the end point values of the range but also intermediate values of the range as explicitly being included within the range and varying by the last significant figure of the range. By way of example, a recited range of from 1 to 4 is intended to include 1-2, 1-3, 2-4, 3-4, and 1-4.
It was discovered that brain proteins from different central nervous system (CNS) cell types are proteolytically broken down after brain injury into PBP and PF. PBP and PF are released from injured tissue into biofluid, typically cerebrospinal fluid and blood. These proteolytic events are brain injury-mediated and are not found in biofluids of subjects that have not had a traumatic brain injury (TBI).
The present invention identifies a multitude of full-length proteins, PBPs and PFs are produced after traumatic brain injury and released into biological fluids. These compounds can be used to identify specific anatomical regions of the brain and subcellular structures affected, and for diagnostic and prognostic tests. The marker PFs and PBPs are identified from fluid biological samples such as cerebrospinal fluid (CSF), serum, plasma or blood samples. Use of methods such as mass spectrometry identifies unique fragments from proteins damaged from traumatic brain injury.
Unique PBPs and PFs are identified which can locate brain damage to specific brain regions such as the cortex, striatum, white matter and the like. Damage can be linked to brain cell types such as neurons, astrocytes, and oligodendrocytes as well as subcellular structures such as axons, dendrites, growth cones, cortical cytoskeleton, intermediate filaments and extracellular matrix.
Brain-specific or specifically brain-enriched proteins from various CNS cell types (including neuron, astrocyte, oligodendrocytes) and extracellular matrix are released and also are proteolytically broken down into PBPs and PFs of large and small sizes as a result of trauma to the central nervous system and are released from the injured tissue into biofluids, such as cerebrospinal fluid and blood, where they can be measured. Since these proteolytic events are brain injury-mediated, these PBP and PF can be used as injury-specific biomarkers, as well as the proteins. This was supported by the identification in the present application of unique PBPs and PFs. The presence and amount of combinations of these markers allows one to determine the presence of damage or injury to specific brain regions, including the cortex, striatum, and white matter, specific brain cell types such as neurons, astrocytes, and oligodendrocytes, and specific subcellular structures, including axons, dendrites, growth cones, cortical cytoskeleton, intermediate filaments and extracellular matrix.
Methods of the invention involve testing fluid biological samples from a subject, such as a mouse traumatic brain injury model or a human central nervous system trauma subject. The sample is subjected to ultrafiltration with a low molecular weight (10,000 Da) cutoff membrane to separate the smaller PFs from the larger PBPs and proteins, then the resulting fractions are subjected to testing to identify specific peptides in the filtrate and the larger peptides and proteins in the retentate. Testing can include a tandem mass spectrometry proteomic method and/or immunological methods such as high sensitivity immunoblotting. Time course measurements of post-injury biofluid levels of these proteins, PBPs, or PFs can be used as TBI and CNS injury diagnostic and prognostic tools at different time periods post-injury when compared to levels as recovery progresses and in normal controls.
A biomarker as defined by the National Academy of Sciences, and as used herein, the presence of which indicates or signals one or more events in biological samples or systems. Biomarkers for central nervous system injury are valuable and unbiased tools in defining the severity of CNS injury because they reflect the extent of brain and spinal cord damage in emergency medicine, neurointensive care and hospitalization settings. The invention therefore includes a fast turn around point-of-care diagnostic biofluid test and device for deployment in various hospital settings. A small amount of subjects' blood samples can be used on the device and levels of specific combination of two or more of the biomarker PFs can be determined.
Generally, the higher the levels of these biomarker levels, the more severe the injury. For example, in an emergency medicine setting, the more severe brain or spinal cord injury subjects can then be admitted to hospital for treatment and monitoring while the mildly injured subjects can be released. Thus the biomarkers of the invention can be used as triaging tools. For subjects already in a neurointensive care unit, unresolved high biofluid levels of CNS biomarkers or further elevations of such biomarkers can indicate the deterioration of the subject's condition or the evolution of the injury. Thus aggressive medical interventions (such as surgery or other procedures or treatments) might be administrated. The PBP and PF biomarkers can be used for monitoring and management of critically injured subjects. For those TBI or spinal cord injury patients who are moderately injured and are staying in hospital, periodic monitoring of their biofluid levels of CNS biomarkers can be useful to detect delayed elevations of the biomarkers, which could indicate occurrence of a secondary injury or the deterioration or evolution of the initially moderate injury to a more severe condition, or development of post-trauma neurodegeneration, allowing more aggressive medical monitoring or medical intervention to be administered. CNS injury biomarkers in the acute or subacute phase can inform on and/or improve neurological recovery or patient outcome. This information can be very useful for patient or caretaker in terms of future care planning, personal life decision-making and arrangement of rehabilitation.
Some metabolite candidates such as N-acetyl aspartate (NAA, a neuronal/axonal marker), creatine (gliosis marker), and choline (indicator of cellular turnover related to both membrane synthesis and degradation) can be used as biomarkers for monitoring the pathobiological changes of primary and secondary damage in TBI using proton magnetic resonance spectroscopy (1H-MRS). In vivo 1H-MRS is a valuable tool for noninvasive monitoring of brain biochemistry by quantifying the changes in the metabolites in brain tissue. However, due to the relatively small size of the spinal cord and magnetic susceptibility effects from the surrounding bony structures, acquiring MR spectra with adequate signal to-noise ratio (SNR) is difficult, and does not allow detection of subtle changes in metabolite levels.
Proteomic analysis is a technique for simultaneously detecting multiple proteins in a biological system. It provides robust methods to study protein abundance, expression patterns, interactions, and subcellular localization in blood, organelle, cell, tissue, organ or organism to provide accurate and comprehensive data about that system. For example, proteomics can use extensive sample procedures and data-dependent acquisition to follow disease-specific proteins (identity and concentration). It facilitates the identification of all differentially expressed proteins at any given time in a proteome (the entire complement of proteins that can be expressed by a cell, tissue, or organism) and correlates and compares these patterns with those in a healthy system during disease progression. Proteomics has been used to study protein expression at the molecular level with a dynamic perspective that helps to understand the mechanisms of the disease.
The complexity, immense size and variability of the neuroproteome and the extensive protein—protein and protein—lipid interactions limit the ability of mass spectrometry to detect all peptides/proteins contained within the sample. Further, some peptides/proteins are extraordinarily resistant to isolation. Therefore, the analytical methods for the separation and identification of peptides/proteins must manage all of these issues. This invention addresses these problems by using separation techniques combined with powerful new mass spectrometry technologies to expand the scope of protein identification, quantitation and characterization.
The complexity of a biological sample can be reduced by separation or fractionation at the protein or peptide level. Multidimensional liquid chromatography (LC) was used in two or more different types of sequential combinations to significantly improve the resolution power and resulted in a large number of proteins being identified. Any of these methods are contemplated for use with the invention.
Ion-exchange chromatography (IEC) in the first dimension was very suitable for the separation of proteins and PFs by separating proteins based on their differences in overall charges. IEC's stationary phase is either an anion or a cation exchanger, prepared by immobilization of positively or negatively charged functional groups on the surface of chromatographic media, respectively. Protein or peptide separation occurs by linear change of the mobile-phase composition (salt concentration or pH) that decreases the interactions of proteins with the stationary phase, resulting in finally eluting the proteins. SDS-PAGE can be used for further protein separation by apparent molecular weight with the resolving distance optimized for the proteome of interest. PFs can be separated by their hydrophobicity using a reversed phase C18 column directly coupled to the electrospray mass spectrometer (ESI-LC-MS/MS). Reversed-phase liquid chromatography (RPLC) is most often used in the second dimension due to its compatibility with downstream mass spectrometry (sample concentration, desalting properties, and volatile solvents).
Mass spectrometry (MS) also is an important tool for protein identification and characterization in proteomics due to the high selectivity and sensitivity of the analysis and can be used in the invention. Electrospray ionization (ESI) is considered a preferred ionization source for protein analysis due to two characteristics: first, the ability to produce multiply-charged ions from large molecules (producing ions of lower m/z that are readily separated by mass analyzers such as quadrupoles and ion traps), and second, the ease of interfacing with chromatographic liquid-phase separation techniques. Electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS) is one of the most commonly used approaches for protein identification and sequence analysis.
This invention takes advantage of proteomic analysis to identify biomarkers in complex biological samples, for example biofluids, to diagnose CNS traumatic injury in a subject, to assess the severity and location of the traumatic injury, and to make a determination of prognosis for the subject. The subject preferably is a human or other mammal, for example a laboratory animal, farm animal, companion animal, zoo animal, or most preferably is a rodent or primate, including a human subject or patient. The mammals contemplated as subjects with respect to this invention include rats, mice, ferrets, swine, monkeys, and primates, including humans.
The injuries contemplated for diagnosis, determination of severity and location, or prognosis include any injury to the central nervous system, of whatever cause. Injuries to the peripheral nerves also are included and are contemplated with respect to this invention. The injury includes injury to the brain, retina, and/or spinal cord, or the peripheral or cranial nerves, and may be localized to a particular physical area or may be generalized. Injuries can be caused by direct trauma, or by inflammation or swelling and edema, contusion, diffuse axonal injury, cerebrovascular injury, hypoxia or anoxia, ischemia, a thromboembolic event, cerebrovascular occlusion or other acute or chronic circulatory disorder, toxins or poisons, envenomation, hemorrhage or hypovolemia, and the like, which cause a physical trauma, directly or indirectly, to the central nervous system. Thus, the subjects referred to herein are any mammal that either suffers from or is suspected of suffering from an injury as discussed above.
The samples that can be usefully collected and tested for protein breakdown products according to the invention include fluid biological samples such as cerebrospinal fluid, whole blood, plasma, serum, and the like, or biopsy, autopsy or necropsy CNS lysate samples and other fluid samples. These samples are collected from the subject according to methods known in the art.
Samples are collected from the subject after an injury to the central nervous system, or an incident that indicates such an injury may have occurred. Incidents such as physical and direct trauma to the head or spine (i.e., sports injury, surgery, vehicular accident, falls, and the like) and its sequelae, illness (i.e., tumor, encephalitis, and the like), or hypoxia (i.e., near drowning, myocardial infarction, embolism, and the like), are specifically contemplated, but are not intended to be limited. The person of skill in the art, such as physician or trauma specialist can easily determine if an injury to the central nervous system is present or should be suspected. Preferably, a sample for diagnostic purposes is collected up to 24 hours after initial injury or up to 3 days (72 hours) after initial injury.
The initial samples can be collected immediately or within about 72 hours after trauma occurs or after injury is suspected, preferably within about 24 hours or one day, and can include one sample only or multiple samples (such as two or more of CSF and blood, serum, brain biopsy, and the like). Further, a second or more than one subsequent sample(s) can be collected at one or several additional subsequent times. For example, samples can be collected hourly, twice daily, daily, every two days, weekly, monthly, or any convenient interval for a period of time deemed to be necessary based on the condition of the patient. A suitable time for continued testing can include two days, a week, two weeks, a month, two months, six months, a year, several years, or for the remainder of a patient's lifetime.
An advantage to collecting multiple samples over a time course (for example, over a week, month, several months, years or longer) is that it allows the practitioner to compare the number, type, and amount of protein breakdown products appearing in the samples over time, to assist in determining the course of the injury or the progress of the subject or patient. Repeated sampling allows the practitioner to determine if peptide levels are diminishing or remaining elevated, thus determining whether the injury to the central nervous system is improving, becoming chronic, or becoming more severe over a course of time.
Intact proteins such as calcium binding protein S100 beta (S100β), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), neuron specific enolase (NSE), neurofilament protein (NFL), SBDP150/SBDP145/SBDP120, ubiquitin C-terminal hydrolase-L1 (UCH-L1) and microtubule-associated 2 (MAP-2) have been identified as potential markers of brain damage. However, due to the complexity of TBI and other central nervous system injury, multiple interventions that target the different complications of the injury may be required in a clinical setting. Previous methods using a single biomarker are unlikely to be successful for either diagnostic or prognostic purposes in human patients. Therefore, although the sample or samples can be tested for only one of the biomarkers disclosed here as part of the invention, it is preferable to test for more than one in each sample. Preferred PFs according to the invention are provided in Table 1, below. In preferred methods, one or two PFs from each protein in the table are tested in each sample. In other embodiments, proteins, PBPs, and/or PFs from each category are analyzed.
The above table shows novel CNS traumatic injury biomarkers identified as PFs derived from CNS proteins due to traumatic injury activated proteolysis, in accordance with the schematic diagram in
Additional TBI proteolytic biomarker PBPs or PFs were also derived from brain proteins Synapsin-I, II, III (SYN1, SYN2, SYN3), Cortexin-1,2,3 (CTXN1, CTXN2, CTXN3), Striatin (STRN), NRGN (fragment), MBPS (fragment) Golli-MBP1, VIM, Brain acidic soluble protein (BASP1, BASP2 (GAP33)), Neurochondrin, Nesprin-1 Glutamate Decarboxylase-1, 2 (GAD1, GAD2), Neurexin-1, 2, 3 (NRXN1, NRXN2, NRXN3) Calmodulin-binding spectrin associated proteins-1, 2, 3 (CAMSAP1, 2, 3), and Chondroitin sulfate proteoglycans (CSPG4, Neurocan (CSPG3) and brevican. These proteins are listed in Table 2, below, showing the brain area in which they are located, and therefore the brain area which is associated with the appearance of the biomarker(s) upon injury. Thus, to determine if an injury to astroglia, for example, is to be diagnosed or investigated, VIM-derived PFs should be analyzed; if an injury to neuron cell bodies is to be diagnosed or investigated, BASP1 and neurochondrin derived PFs should be analyzed.
The above table provides proteins or proteolytic PFs released after traumatic injury to the CNS (e.g. TBI) and their associated brain region, brain cell type or neuronal subcellular location. The work presented here used an in vitro brain injury model with mouse brain lysate and purified brain protein incubation with calcium solution or protease calpain, an in vivo mouse traumatic brain injury model and human traumatic brain injury biofluid (cerebrospinal fluid or CSF) samples. These samples were analyzed using separation by ultrafiltration with low a molecular cutoff filter, a tandem mass spectrometry proteomic method and immunological methods including high sensitivity immunoblotting to detect and identify a number of brain-specific or brain-enriched proteins from various CNS cell types (neurons, astrocytes, oligodendrocytes) or extracellular matrix. Proteins in the central nervous system are proteolytically broken down into PBPs and PFs upon injury to the tissues. The PBPs and PFs are released from injured tissue into biofluids (such as cerebrospinal fluid and blood) and can be detected there as shown above. Since these proteolytic events are brain injury-mediated, the PBPs and PFs were identified to be injury-specific biomarkers.
The proteins, PBPs, and PFs described here are identified in a sample from a subject such as a human patient who has suffered an injury to the central nervous system or who is suspected of having suffered such an injury. Preferably, a sample is obtained from the subject within 24 hours of the injury or suspected injury. A series of samples also can be taken over a period of days or weeks so that progress can be determined. The sample preferably is CSF or whole blood/serum. Secondary preferred samples are saliva, urine, nasal fluid and tears.
In the case of diagnosing an acute injury or suspected acute injury, a first sample is taken after the injury, preferably as soon as possible and within 24 hours, and further samples can be taken over a time course to obtain information on continued injury or recovery. Testing can be performed to detect a single protein, PBP, or PF, or a combination of one or more proteins, PBPs, or PFs. In some inventive embodiments, at least one protein, PBP, or PF for each of the injury types in Table 1, above, is tested. A high level of one or more of these (approximately twice the level as found in a control sample or uninjured subject or more) indicates an injury, and the identity of the peptide indicates the particular area that has been injured. A peptide level of about 1.5-2.5 times higher than control, or 2.0-2.5 times higher than control (for example about 1.5, 1.75, 2, 2.25, or 2.5 times higher than control), indicates a mild injury; a peptide level of about 2.5-4.0 times higher than control (for example about 2.5, 2.75, 3.0, 3.25, 3.5, 3.75 or 4 times higher than control) indicates a moderate injury; a peptide level of more than about 4.0 times higher than control (for example 4.25, 4.5, 4.75, 5, 5.25, 5.5, 6, 6.5, 7 or more) indicates a severe injury, with amounts higher than 6 times higher than control indicating a very severe injury.
In the case of diagnosing a chronic injury or a suspected chronic injury, a series of samples are taken periodically so that the results can be compared along a time course as well as compared to a control sample from an uninjured subject or an in vitro sample produced for that purpose. Analyte (protein, PBP, or PF) levels that increase over time indicate a chronic or worsening injury; analyte levels that remain about the same over time indicate a stable state or chronic injury; analyte levels that decrease over time indicate that the injury is improving or is not continuing. The levels for determining the severity of the chronic injury are the same as those discussed above for an acute injury.
The precise testing of the samples to be performed to make a diagnosis can be determined by the routine practitioner, depending on the condition of the patient and the suspected type and severity of the injury. For example, if a particular injury to a brain area or subcellular area is suspected after examination of the subject, the sample can be tested for breakdown products derived from the protein identified as correlating with that particular area in this application so that the diagnosis can be confirmed. If the injury is unknown, a large number of tests or the entire panel of tests for all breakdown products can be performed on the sample to make a specific diagnosis.
A diagnosis of a particular injury is made by comparing the results of a subject sample to an uninjured control. If the subject sample has a significantly higher amount of the diagnostic protein, PBP, or PF than the control, a positive diagnosis can be made.
To determine the severity of an injury or prognosis for the subject, the level of a protein, PBP, or PF, or a battery of proteins, PBPs, and PFs can be compared to control samples of varying injury. For example, higher biofluid levels of one or more of the analytes can be correlated to the severity of traumatic injury, to the likelihood of development of post-trauma complications, or to a poor patient prognosis.
The invention contemplates kits for testing for brain protein breakdown products as described herein, and can include, for example, one or more of the following: suitable containers and equipment for obtaining a subject sample such as CSF or blood; ultrafiltration cell(s) or units with a molecular weight cutoff of about 10 kDa; one or more antibodies or aptamers that specifically recognize a protein, PBP, or PF according to the invention as described herein; and protein, PBPs, and/or PFs according to the invention as described herein to be used as standards in assays. Alternatively, if a mass spectrometry method is to be used for analyte detection, the kit can include analyte standards to be used as internal standards (spike in) or external standards (side-on-side).
A kit according to the invention comprises components for detecting and/or measuring the breakdown products described herein in a sample from a subject. Preferably, the kit contains a primary antibody or aptamer reagent or reagents that each specifically bind to a peptide breakdown product. The antibodies or aptamers can be organized into groups of reagents that recognize the breakdown products of a single protein or a group of proteins that indicate a certain type of central nervous system injury, if desired. Also, the antibodies or aptamers can be organized into panels of reagents that together can detect the breakdown of some or all of the indicator proteins identified here.
The primary antibodies (preferably monoclonal antibodies or fragments thereof) or aptamers specifically recognize and bind to a single peptide or class of peptides. One or more secondary antibodies (optionally labeled) that bind to the primary antibody or aptamer also can be included, as well as a target antigen (the peptide to be detected in the sample). The secondary antibodies can be, for example, antibodies directed toward the constant region of the primary antibody (optionally IgG) (e.g., rabbit anti-human IgG antibody), which may itself be detectably labeled {e.g., with a radioactive, fluorescent, colorimetric or enzyme label), or which may be detected by a labeled tertiary antibody {e.g., goat anti-rabbit antibody).
The antibody- or aptamer-based detection methods can involve a western blot, immunoassays such as enzyme linked immunosorbant assays (ELISA), sandwich assay, or radioimmunoassay (RIA), mass spectrometry, or antibodies or aptamers can be used in combination with mass spectrometry detection methods (e.g., LC-MS/MS). Any detection assay method for proteins and/or peptides known in the art can be used. Suitable containers for performing the assays also can be included in a kit for convenience. Such assays are well known in the art, and any of these known methods can be used with the invention to detect PBP or PF according to the invention. In certain embodiments of the invention, a fast turn around point-of-care diagnostic biofluid test and device can be deployed in various hospital settings. The test will use a biochip or cartridge that contains one or two biomarker target-specific capture and detection antibodies or aptamers. The POC device ha s receptacle for the biochip or cartridge as well as a part that can generate a readout signal. Commonly for these detection methods, the biomarker readout is in the form of light, chemiluminescence or fluorescence signals, chemoelectric signals, radiation signal or absorbance signals. However, mass spectrometry and tandem mass spectrometry methods might also be employed.
A diagnostic test kit generally includes a cartridge or biochip with embedded capature and/or detecting agents (e.g specific antibodies) for one or more protein, PBP. and/or PF biomarker, along with a companion reader or analyzer with a receptacle for the detection cartridge as well as a component capable of producing a biomarker readout. Alternatively a detection kit can be a sandwich ELISA (with capture and detection antibodies for each biomarker) in a singlet or multiplex fashion, as it is commonly described in the field of diagnostics. The detection kit also can be an immunoblotting or western blotting format, as it is commonly described in the field of biochemistry and diagnostics. The common readout from the above mentioned test kits is in the form of light signals (e.g. fluorescence, chemiluminescence), absorbance changes or electrochemical signals. However, mass spectrometry and tandem mass spectrometry methods might also be employed.
Preferably, instructions are packaged with the other components of the kits of the invention, for example, a pamphlet or package label. The instructions explain how to perform testing and methods according to the invention.
In some embodiments, a diagnostic kit comprises (a) detection agents for antibody, aptamer or mass spectrometry detection methods for detection of one or more PFs or other analytes, (b) an analyte protein, protein breakdown product, or PF to serve as internal standard and/or positive control; and (c) a signal generation coupling component. Such signal generation components either are based on detection tool (e.g. antibody) coupled enzyme, which carries out enzymatic reaction to generate a product or direct coupled of a tagging molecule to the detection tool (e.g. antibodies). These enzymatic protein or ragging molecules generally product a light, fluorescence, or chemiluminescence signal, or absorbance changes or electrochemical signals, or the like, to allow detection. However, mass spectrometry and tandem mass spectrometry methods might also be employed.
This invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined, otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein, are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The following examples are provided as illustrations of the invention and are in no way to be considered limiting.
1. Sample Collection and Preparation
Brain samples from CB57BL/6 male mice, 3 to 4 months old, were used. Cortex, corpus callosum and hippocampus regions were isolated from each mouse brain. The brain samples were pulverized to powder using mortar and pestle placed over dry ice to maintain a cold environment. The pulverized brain samples were then lysed using Triton lysis buffer (20 mM Tris-CHl, 5 mM EGTA, 100 mM NaCl, with 1% Triton) by incubating at 4° C. for 90 minutes. After incubation, the samples were centrifuged and a protein assay was performed to estimate the concentration of the mouse brain lysates. Brain lysate equivalent to 120 μg of protein was used.
For some samples, purified protein (GFAP, MBP, NRGN (2-10 ug)), or brain lysate (50-160 ug) were subjected to in vitro incubation with 7 mM calcium chloride CaCl2) or with calcium and human calpain-1 protease (protease: brain protein ratio of 1:20 to 1:50) and 20 mM (NH4)2CO3, 10 mM dithiothreitol (DTT) and 7 mM CaCl2) (pH 7.4). This condition mimics the brain injury induced calpain activation in animal and human brain, and serves as an in vitro model of central nervous system injury.
Centrifuged CSF samples (500 uL) were obtained from human subjects with severe TBI (Glasgow coma score of 3-8) and from control, uninjured subjects.
Ultrafiltration was used to separate smaller from larger peptide molecules. The brain lysate and the CSF samples were filtered through 10,000 Da molecular weight cutoff membrane filters (Sartorius Stedim Biotech®, Goettingen, Germany). This filtration technique allows the isolation in the ultrafiltrate of molecules that are smaller than or equal to 10,000 Da, from the retentate.
The ultrafiltrate then was concentrated using a vacuum evaporation method (SpeedVac™; (Thermo Scientific®) to a volume of 5 μL. The concentrated samples were reconstituted with water containing 0.1% formic acid. These samples were ready for liquid chromatography-tandem mass spectrometry. The samples of retentate of ultrafiltration were analyzed using western immunoblotting methods.
Tandem mass spectrometry-based proteomic methods first were used to identify PFs derived from the brain injury protein biomarkers using in vitro calcium or calpain digestion of purified protein or TBI-model mouse brain lysate. The samples were analyzed using a system with a Thermo Scientific® LTQ-XL (Thermo Fisher Scientific®, San Jose, Calif., USA) with a Waters® nanoACQUITY UPLC system ((Waters®, Milford, Mass., USA). LC-MS grade water and acetonitrile, both with 0.1% formic acid, were used as mobile phases with a 115 minute gradient at a flow rate of 300 nL/min on a 1.7 μm BEH130 C18 column (100 μm×100 mm). Tandem mass spectra with data-dependent acquisition (top 10 most abundant ions) method was performed using Xcalibur® 2.0.7 (Thermo®). MS/MS data were searched using Proteome Discoverer® 1.3 (Thermo®) against mouse database and human database respectively with no enzyme.
Western blot was performed on the higher molecular weight proteins (greater than about 10,100 Da) that were retained on the membrane filter. Western blot was used to confirm proteolysis of proteins in the CCI and TBI samples. SDS-gel electrophoresis and immunoblotting was done using standard published methods (see Yang et al., PLOS ONE 5, e15878, 2010). Blotting membrane was probed with specific target-based antibody (1/500 to 1/2,000 dilution) followed by secondary anti-mouse or anti-rabbit HRP (horse radish peroxidase) conjugate antibody and then detected visually using 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium (NBT/BCIP) as substrate (colorimetric development). Other immunological assays, such as ELISA (i.e., sandwich assays), RIA, and others known in the art can be used to detect and quantitate the proteins, PBPs and PFs according to the invention, as is convenient to the practitioner. In general, immunological assays such as a sandwich ELISA are preferable for detection of larger peptides and proteins.
In order to produce an in vivo model of traumatic brain injury in mice, a controlled cortical impact (CCI) device was used according to known methods (see Yang et al., J. Cerebral Blood Flow Metab. 34:1444-1452, 2014). CB57BL/6 mice (male, 3 to 4 months old, Charles River Laboratories®, Raleigh, N.C., USA) were anesthetized with 4% isoflurane in oxygen as a carrier gas for 4 minutes followed by maintenance anesthesia of 2% to 3% isoflurane. After reaching a deep plane of anesthesia, mice were mounted in a stereotactic frame in a prone position, and secured by ear and incisor bars. A midline cranial incision was made and a unilateral (ipsilateral) craniotomy (3 mm diameter) was performed adjacent to the central suture, midway between the bregma and the lambda. The dura mater was kept intact over the cortex. Brain trauma was induced using a PSI TBI-0310 Impactor (Precision Systems and Instrumentation®, LLC, Natick, Mass., USA) by impacting the right cortex (ipsilateral cortex) with 2 mm diameter impactor tip at a velocity of 3.5 m/second, 1.5 mm compression depth, and a 200 millisecond dwell time (compression duration). Sham-injured control animals underwent identical surgical procedures but did not receive an impact injury. Naïve animals underwent no procedure.
See
From the list of mass spectrometry data on PFs obtained using database searches and complimentary immunblotting evidence from protein digestions on samples from a mouse model of TBI and human central nervous system injured subject biofluid samples (based on the peptide XCorr valuses (e.g., XCorr >3.0), brain cell type specific markers identified in this application include the proteins in Table 2, above. See also
Mice subjected to traumatic brain injury as described in Example 2 were sacrificed. Cortex and hippocampus tissue sample lysates were subjected to ultrafiltration and the ultrafiltrates tested by nLC-MSMS to identify TBI-induced PFs. The PFs were identified by comparison with immunoblotting data on proteins/PBPs. Results are shown in Table 3, below. The data showed that the in vitro incubation model and the mouse model of TBI both resulted in production of similar brain PBPs/PFs than those found in the CSF samples of human TBI subjects. PBPs or PFs identified by all three methods therefore can have utility in diagnosing or monitoring human brain damage.
1213.31
1194.30
1170.62
1132.61
1108.93
1085.25
1061.57
1037.89
1292.68
1219.14
1141.09
1112.58
1044.05
1343.65
1215.55
1128.52
1071.50
866.45
828.10
1184.14
789.76
1127.59
1013.54
605.98
568.29
1062.61
331.19
NGRN (NP_071312) PFs identified in TBI mice brain lysate ultrafiltrate samples are given in
1874.33
1790.83
1762.32
1466.75
1331.19
1152.60
1021.53
1941.01
1839.97
1651.89
1495.79
1394.74
1144.64
1188.9
1151.2
1108.2
1052.6
1014.9
1416.7
1367.2
1043.1
1360.7
1031.5
1092.28
455.58
556.68
327.40
911.09
456.05
504.61
553.17
509.53749
The peptides identified in this Example show the distinct PFs released into the fluid biological sample ultrafiltrate of in vitro calpain proteolyzed human GFAP protein. This method mimics the human TBI conditions where calpain is known to be hyperactivated and to attack cellular proteins in the brain.
Table 11 shows the GFAP PFs identified in ultrafiltrate samples from a calpain-digested sample of purified human GFAP protein. The calpain proteolysis mimics CNS traumatic injury-induced calpain activation. A number of GFAP PFs were identified, as shown in Table 11, below.
The sequence of human GFAP (Accession No. P14136; 432 amino acids; GI:251802) is as below (regions with GFAP PFs identified are shown in bold).
EGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM
Since GFAP is a major astrogial protein that is also involved in post-injury gliosis (glia cell hypertrophy and proliferation), the release of GFAP PFs can indicate astroglia cell injury. Thus, this example shows that biofluid-based monitoring of the GFAP-released PFs can be used to monitor astroglia injury mediated by calpain activation.
Table 11 shows Tau PFs, generated by calpain digestion of Tau-441 protein and are found in ultrafiltrate samples. Tau-441 PFs generated by calpain digestion (mimicking TBI) include Tau N-terminal region peptide 1 AEPRQEFEVMEDHAGTYGLG (aa 2-21; SEQ ID NO:249); Tau N-terminal region peptide 2AAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVS (aa 90-123; SEQ NO:250); Tau center region peptide LSKVTSKCGSLG (aa 315-326; SEQ ID NO:251); Tau C-terminal region peptide 1 SPRHLSNVSSTGSIDMVDSPQLA (aa 404-426; SEQ ID NO:252); and Tau C-terminal region peptide 2 TLADEVSASLAKQGL (aa 427-441; SEQ ID NO:253). Table 11 lists further PFs along with MS/MS data for PFs found in TBI subject CSF ultrafiltrate samples or derived from in vitro calpain digestion of Tau and phospho-Tau protein (Tau-441; a model that mimics CNS traumatic injury-induced calpain activation).
The sequence of human Tau-441 (microtubule-associated protein Tau isoform 2; P10636-8) is:
TTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTK
GSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPM
GDTSPRHLSNVSSTGSIDMV DSPQLATLADEVSASLAKQGL.
The Tau PFs identified are shown in bold. Key Tau PFs identified here are shown in Table 12, below.
265.62647
315.16068
494.25330
1058.53643
529.77185
1186.63139
593.81934
1314.68997
657.84862
1371.71143
686.35936
Table 15, below shows the origin of PBP and PF biomarkers derived from additional proteins in mouse cortex or hippocampal ultrafiltrate samples after TBI (day 1 to day 3 post-injury. This example further supports use of biofluid-based monitoring of either specific brain protein PBPs or their unique PFs to inform on different brain vulnerabilities after brain injury (i.e., axonal marker astroglia, myelin and presynaptic terminal damage, respectively).
813.26
930.09
775.24
756.39
888.06
865.44
721.37
842.44
702.20
819.43
683.03
800.02
666.85
788.62
657.35
751.60
626.50
737.39
723.19
669.15
557.79
620.92
606.72
427.40
501.28
The NGRN PFs included those listed in Table 17, below. The full sequence of NRGN (78 amino acids) is
KSGERGRKGPGPGGPGGAGVARGGAGGGPSGD.
Underlined residues show the area in the sequence where PFs are produced.
Table 17 is a representation showing the NRGN-derived PFs generated and released into CSF from human TBI subjects. Duplicate PFs found are not shown. None of the PFs shown was found in non-injured control CSF samples.
VIM PFs Identified from human TBI subjects also were characterized.
592.79
528.74
463.22
427.70
371.16
313.64
257.10
192.58
100.52
844.40294
1209.55874
605.28301
1337.66133
1240.60856
414.20771
The amino acid sequence of human VIM (accession #P08670) is:
MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRP
STSRSLYASSPGGVYATRSSAVRLRSSV
PGVRLLQDSVDFSLADAI
LPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVIN
ETSQ
Residues underlined and in bold show the areas which the VIM PFs are released.
Preferred PFs according to the invention include those listed in Table 20 below and in
MSTRSVSSSS YRRMFGGPGT ASRPSSSRSY
VTTSTRTYSL GSALRPSTSR SLYASSPGGV
YATRSSAVRL RSSVP
MBP PFs were identified.
230.24
345.33
957.03
474.45
841.94
712.83
598.72
501.61
883.93
402.47
511.04
303.34
The full sequence of human MBP isoform 1 (classic MBP, 21 kDa, 197 amino acids, (NP_001020252.1) is:
SRF
SWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLG
Underlined and bold residues show the areas where PFs originate.
1245.40
1188.35
594.68
551.14
487.05
The location of the peptide within the N-terminal region of human MBP Isoform 3 (197 aa) accession #167P02686-3 is shown in the sequence (underlined and bold):
Additional sequences within this MBP isoform include PRHRDTGILDSIGR; SEQ ID NO:328, GRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRF; SEQ ID NO:329, and HKGFKGVDAQGTLS; SEQ ID NO:330.
667.50
550.04
382.38
1562.75
1306.56
262.12
The full sequence of human Golli-MBP1, accession #P02686 (304 aa; 34 kDa), is:
HPADPGSRPH LIRLFSRDAP GREDNTFKDR PSESDELQTI
GFL
PRHRDTG ILDSIGR
FFG GDRGAPKRGS GKDSHHPART
AHYGSLPQKS H
GRTQDENPV VHFFKNIVTP RTPPPSQGKG
RGLSLSRF
SW GAEGQRPGFG YGGRASDYKS A
HKGFKGVDA
QGTLS
KIFKL GGRDSRSGSP MARR.
The italic sequence in Golli-MBP isoform 1 above is identical to that of human MBP isoform 5 (#P02686-5, 171 aa).
Golli-MBP isoform 1 PFs found in human TBI CSF ultrafiltrate samples are of the following sequences: residues 4-34 of this Golli-MBP isoform 1 sequence as HAGKRELNAEKASTNSETNRGESEKKRNLGE (SEQ ID NO:334); residues 75-116 of this sequence as NAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDE (SEQ ID NO:335). These two peptide unique fragments are derived from the N-terminal region of Golli-MBP isoform 1, and are not found in classical MBP isoform 5. Additional Golli-MBP isoform 1 PFs found in human TBI CSF ultrafiltrate samples are of the following sequences: residues 144-157 of this sequence of Golli-MBP1, accession #P02686 (304 aa) as HGSKYLATASTMDH (SEQ ID NO:336); residues 164-177 as PRHRDTGILDSIGR (SEQ ID NO:337; residues 212-248 as GRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRF (SEQ ID NO:338); and residues 272-285 as HKGFKGVDAQGTLS (SEQ ID NO:339). These sequences are found in both the Golli-MBP isoform and classical MBP isoform 5. These PF sequences in the Golli-MBP isoform 1 sequence are shown as underlined (see above SEQ ID NO:340aa).
The full sequence of human MBP isoform 5; #P02686-5; 171 aa; 18.5 kDa) is:
PPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGT
Underlined sequences are MBP PFs identified in human TBI CSF ultrafiltrate samples as shown above.
Table 23, below, summarizes MBP PFs found in human TBI CSF that are derived from both human Golli-MBP1 (304 aa, #P02686-1) and MBP Isoform 3 ((171 aa; #P02686-5). The sequences of human Golli-MBP1 (SEQ ID NO:342aa) and classic MBP Isoform 3 (SEQ ID NO:343bb) are shown. The common regions of both isoforms are in italics. PFs derived from a distinct N-terminal region identified in Golli-MBP1 (SEQ ID NO:344aa) are shown in italics. This example further shows that biofluid-based monitoring of classic MBP (e.g., MBP3, MBP5) and Golli-MBP1 fragments or peptides can be used to monitor oligodendrocyte/myelin damage/white matter injury.
Table 23 presents selected PFs detected in human CSF samples from TBI subjects. See Table 24, below.
1730.28051
1605.23283
1472.19945
1604.84546
1023.51691
1241.10873
596.29090
710.04198
532.78330
377.72585
1337.77604
1112.66470
761.40396
575.33990
476.27149
363.18743
235.09246
Overall, these data indicate that a number of GFAP PFs in the GFAP alpha isoform (#P14136; 432 aa) are found in TBI CSF samples and can serve as biomarkers for TBI or traumatic injury to the CNS. This example also shows that human biofluid-based monitoring of PFs of GFAP can be used to monitor astroglia injury. See Table 30, below for a list of selected peptides.
The full sequence of glial fibrillary acidic protein (human) alpha isoform (#P14136; GI:251802 (with the regions where the PFs occur shown in bold) is:
GHLKRNIVVKTVEMRDGEVIKESKQEHKDVM.
RSYVSSGEMMVGGLAPGRRLGP
Table 32, below provides a list of PFs showing an isoform specific peptide for the high molecular weight Tau-758 (identifier: P10636-19; 776aa). These PFs can be detected in TBI CSF samples, but in not control CSF.
SPKHPTPGSSDPLIQPSSPAVCPEPPSSPKYVSSVTSRTGSSGAKEM
The following tables show the sequences of Tau-441 and Tau-758. The isoform unique sequences are underlined. The Tau-758 PFs found in human TBI CSF ultrafiltrate are shown in bold. See Table 33 and Table 34, below.
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYT
MHQDQEGDTDAGLKESPLQT
GSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPM
MSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRHAPELLKHQLLGDLHQE
GPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTA
AREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQDAPL
EFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEA
DLPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRS
SAKTLKNRPCLSPKHPTPGSSDPLIQPSSPAVCPEPPSSPKYVSSVTSRT
GSSGAKEMKL
]KGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKT
Table 35, below, provides a summary of MS/MS results on PFs identified from Tau protein isoforms Tau-758 and Tau-441 in human TBI CSF ultrafiltrate samples.
This example shows that human biofluid-based monitoring of Tau-F (Tau-441) and Tau-G (766 aa) and its PBPs or PFs can be used to monitor axonal injury or neurodegeneration.
In addition,
The presence of proteolytic breakdown products of CAMSAP3 in TBI CSF implies that CAMSAP1 protein and its higher molecular weight breakdown products are present and in higher in biofluids (CSF) from TBI subjects than in controls. This example therefore shows that human biofluid-based monitoring of CAMSAP1 and CAMSAP3 PBPs or PFs can be used to monitor axonal damage.
The presence of PFs of MAP6 in TBI CSF indicates that MAP6 protein and its higher molecular weight breakdown products are present and higher in biofluids (CSF) from TBI subjects than in controls. This example shows that human biofluid-based monitoring of MAP6 PFs can be used to monitor dendritic injury.
The sequence of microtubule-associated protein 6 (human) (Q96JE9-1) is:
QQAQPALAPPSARAVAIETQPAQGELDAVARATGPAPGPTGEREPAAGPG
PTAPHTEYIESSP.
Regions in bold are MAP6 PFs found in human TBI CSF ultrafiltrate samples.
Complment protein Breakdown Products and Peptide Fragments. As shown in Table 48A, Complement protein Clqb, C3, C5, Cls, and CR1 peptides were identified in only human CSF samples, not control CSF samples.
Table 49, below, is a spreadsheet showing additional representative PFs from brain proteins uniquely identified from human CSF ultrafiltrate samples. Table 50, below, shows combined evidence of PFs from brain proteins (peptidome) found in brain ultrafiltrate in the mouse model of TBI and/or in CSF samples from human TBI subjects. This summarizes the results showing that human biofluid-based monitoring of additional brain protein derived PFs can be used to monitor central nervous system injury such as TBI.
GPGGPGGAGVARGGA
GGGP
1Modification: C19(Carbamidomethyl); M22(Oxidation).
2Modification: M13(Oxidation).
3Modification: C6(Carbamidomethyl); M8(Oxidation); M12(Oxidation)
4Modification: C7(Carbamidomethyl)
5Modification: M5(Oxidation); M29(Oxidation)
6Modification: M18(Oxidation)
7Modification: M8(Oxidation); M19(Oxidation)
8Modification: M22(Oxidation)
9Modification: M9(Oxidation); M13(Oxidation); M27(Oxidation)
Additional key novel TBI PBP biomarkers identified were derived from Synapsin-I, II, III (SYN1, SYN2, SYN3), Cortexin-1,2,3 (CTXN1, CTXN2, CTXN3), Striatin (STRN), NRGN, Golli-MBP1, Tau-758, VIM, Brain acidic soluble protein (BASP1, BASP2 (GAP33)), Nesprin-1, Glutamate Decarboxylase-1, 2 (GAD1, GAD2), Neurexin-1, 2, 3 (NRXN1, NRXN2, NRXN3) Calmodulin-binding spectrin associated proteins-1, 2, 3 (CAMSAP1, 2, 3), and Chondroitin sulfate proteoglycans (CSPG4, Neurocan (CSPG3, brevican), and Neurochondrin. These proteins are listed in Table 48, with supporting data in Table 15. This example shows that human biofluid-based monitoring of additional these brain protein derived PBPs and/or PFs can be used to monitor brain injury such as TBI.
For diagnosis, prognosis or monitoring of trauma to the central nervous system the biofluid levels of protein, PBPs and PFs, or a battery of proteins, PBPs and/or PFs are measured. An initial subject fluid biological sample (such as blood, serum, plasma or CSF) is obtained within 24 or 72 hours after traumatic injury or suspected traumatic injury to the CNS (such as TBI), preferably within 24 hours after traumatic injury. The sample is subjected to ultrafiltration with a molecular cutoff of 10,000 Da, using a centrifugation-based ultrafiltration cell. The retentate is subjected to protein analysis. The filtrate is subjected to testing for PFs using an antibody-based immunoassay according to procedures well-known in the art, using antibodies that specifically recognize AEPRQEFEVMEDHAGTYGLG (SEQ ID NO:465), NVKMALDIEIAT (SEQ ID NO:466), DGEVIKES (SEQ ID NO:467), and GRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRF (SEQ ID NO:468). The signal indicating the amount of the peptide is compared to the signal from an equivalent control sample from a control, uninjured subject. An amount of one or more PFs that is two times the control amount, indicates an injury. Sample interpretations of results are shown in Table 51.
In order to determine the prognosis of the subject above, the following further tests should be performed on samples collected from the subject at the following times: 24 hours, 48 hours and 72 hours post injury. If the 72-hour results are less than ⅓ of the levels for the 24-hour results, the prognosis is good to excellent; if the 72-hour biomarker test levels are about the same as or higher than the levels seen in the sample taken at 24 hours, the prognosis is poor.
For novel Golli-MBP protein,
By comparing the signals yielded for specific proteins, PBPs and/or PFs to available standards (such as cranial/spinal computer tomography (CTO or Magnetic resonance imaging (MRI) detectable abnormality or Glasgow coma scale score, or Glasgow outcome scale score), their cutoff values can be assigned. Such cutoff values are compared to control samples or to a prepared chart of levels to determine the severity of the injury, or the prognosis of the subject, or monitoring of the patient injury progression or recovery. For example, higher biofluid levels of one or more protein, PBP or PF indicates the subject is more severely injured, more likely to develop post-trauma complications, or to prone to have poor patient outcome. For example, for blood levels of a protein, PBP or PF (e.g. as derived from synapsin) usually would have levels in control subjects of less than 10 pg/mL, while mild to moderate CNS injured subjects generally are expected to have a level between 10-50 pg/mL, and more severe CNS injury subjects generally are expected to have a level above 50 pg/mL
In another example, at least two measurements of these proteins, PBPs, and PFs as biomarkers are assayed in an initial and at least one subsequent sample. For example, first measurement within 24 hours of the incident, and a second or additional measurement after the first 24 hours. The values of these biomarker levels over time provide the ability to monitor the progression of the traumatic injury or the recovery of the CNS from the initial traumatic injury. For example, a CNS trauma subject that is on course for good recovery with no complications would have biomarker levels in the second or additional measurements that are lower than the biomarker levels of the same biomarker(s) at a prior measurement. On the other hand, a subject who has biomarker(s) levels in the second or additional measurements that are higher than the biomarker levels of the same biomarker(s) at a prior measurement could indicate there is a deterioration or evolution of the injury condition, development secondary injury or post-trauma neurodegeneration development. For this later group, once identified, more aggressive medical monitoring and/or medical intervention then can be administrated.
References listed below and throughout the specification are hereby incorporated by reference in their entirety.
This invention was made with Government support under Contract No. R21 NS085455-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/047030 | 8/19/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62719254 | Aug 2018 | US |
