WO2008008819A2 - Diagnosis and treatment of neurological inflammation - Google Patents

Diagnosis and treatment of neurological inflammation Download PDF

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Publication number
WO2008008819A2
WO2008008819A2 PCT/US2007/073233 US2007073233W WO2008008819A2 WO 2008008819 A2 WO2008008819 A2 WO 2008008819A2 US 2007073233 W US2007073233 W US 2007073233W WO 2008008819 A2 WO2008008819 A2 WO 2008008819A2
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Prior art keywords
cytokines
mip
mcp
protein
injury
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PCT/US2007/073233
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French (fr)
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WO2008008819A3 (en
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Andrew K. Ottens
Mark S. Gold
Firas H. Kobeissy
Kevin Kw Wang
Ronald L. Hayes
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University Of Florida Research Foundation, Inc.
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Publication of WO2008008819A2 publication Critical patent/WO2008008819A2/en
Publication of WO2008008819A3 publication Critical patent/WO2008008819A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/40—Disorders due to exposure to physical agents, e.g. heat disorders, motion sickness, radiation injuries, altitude sickness, decompression illness
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention provides for the detection and diagnosis of chemical neurological injury. Detection of cytokines allows for methods of treating and monitoring the course of treatment in patients with neural injury.
  • Methamphetamine also known as ice, crank, speed, crystal meth is an addictive psycho stimulant belonging to the amphetamine family, which was originally introduced as an appetite suppressant and a treatment for attention deficit disorder. It is the most widely spread illicit drug because it is easy to prepare and cheap to obtain. Methamphetamine can be smoked, inhaled, or injected leading to a variety of social behaviors with a feeling of euphoria and increased self esteem, with the effects lasting up to 15 hours.
  • Amphetamines are sympathomemetics that act by increasing the amount of dopamine, norepinephrine, and epinephrine neurotransmitters available in the brain.
  • the user may experience a heightened sense of awareness and paranoia.
  • the average methamphetamine dose may cause the user to experience mydriasis (dilated pupils), hypertension, tachycardia (increased heart rate), and hyperthermia.
  • Other indicators of methamphetamine use include auditory hallucination, visual hallucinations often involving bugs, agitation, insomnia, anxiety, nausea, and vomiting.
  • Upon methamphetamine overdose, psychosis, myocardial infarction, seizures, and even death may occur.
  • Methamphetamine is a psychostimulant that is known to mediate addictive behavior by acting on the monoaminergic system leading to an increase in the dopamine (DA) and serotonin (5-UT) levels in certain brain regions.
  • DA dopamine
  • 5-UT serotonin
  • methamphetamine use induces dopaminergic and serotonergic axonal terminal damage that is coupled with neuronal degeneration of specific population of neocortical neurons, and is attributed to the lipophilic nature of methamphetamine, which facilitates crossing of the blood brain barrier (BBB) and access to different brain regions.
  • BBB blood brain barrier
  • meth Upon entering the monoaminergic system, meth binds to the plasmalemal dopamine transporter (DAT) and alters its function by blocking the re -uptake of DA and overloading the synapse.
  • DAT plasmalemal dopamine transporter
  • Methamphetamine also diffuses via the DAT into the neuronal terminal where it acts as a substrate for a number of neuronal structures, including the dopamine vesicular transporter (VMAT) that contributes to increased cytosolic DA. Afterward, neuronal function is altered leading to a reduction in DAT activity and the dysfunction of tyrosine hydroxylase (TH) and dopamine vesicular transporter. Furthermore, the oxidizing environment of the cytosol can lead to DA oxidation leading to the generation of nitrogen, oxygen, and metabolic reactive species that trigger dopaminergic terminal degeneration and subsequently cause necrotic cell death.
  • VMAT dopamine vesicular transporter
  • TH tyrosine hydroxylase
  • necrotic cell death the oxidizing environment of the cytosol can lead to DA oxidation leading to the generation of nitrogen, oxygen, and metabolic reactive species that trigger dopaminergic terminal degeneration and subsequently cause necrotic cell death.
  • methamphetamine can diffuse into other neuronal organelles, such as the mitochondria where it causes perturbation in the mitochondrial electron gradient leading to mitochondrial mediated apoptotic cell death.
  • Oxidative stress mediated injury is exacerbated by increased glutamate levels, which in turn can activate NMDA receptors producing more reactive oxygen species leading toward excitotoxicity.
  • Neuronal degeneration is not confined to the monoaminergic system but can include other neocortical cells in a dopamine independent pathway.
  • An immortalized neuronal cell line when treated with methamphetamine exhibited an apoptotic cell death phenotype.
  • a method of diagnosing neurological injury in a patient comprises identifying cytokines and cytokine levels in a sample from an injured patient, wherein the cytokines detected are elevated or decreased as compared to normal individuals which can reflect on the altered immune status of the inflicted patient.
  • the cytokines detected comprise: IL-I, IL- l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ .
  • the cytokine levels can be correlated to known biomarkers of neurological injury.
  • neural proteins such as for example, axonal proteins - NF-200 (NF-H), NF-160 (NF-M), NF-68 (NF-L); amyloid precursor protein; dendritic proteins - alpha-tubulin (P02551), beta-tubulin (PO 4691), MAP- 2A/B, MAP-2C, Tau, Dynamin-1 (P21575), Dynactin (Q13561), P24; somal proteins - UCH- Ll (Q00981), PEBP (P31044), NSE (P07323), Thy 1.1, Prion, Huntington; presynaptic proteins - synapsin-1, synapsin-2, alpha-synuclein (p37377), beta-synuclein (Q63754), GAP43, synaptophysin, synaptotagmin (P21707), syntaxin; post-synaptic proteins - PSD95, PSD95, PSD95, PSD95,
  • alpha-adrenoreceptor subtypes e.g. (alpha (2c)
  • GABA receptors e.g. GABA(B)
  • metabotropic glutamate receptor e.g. mGluR3
  • NMDA receptor subunits e.g. NR1A2B
  • Glutamate receptor subunits e.g. GluR4
  • 5-HT serotonin receptors e.g. 5- HT(3)
  • dopamine receptors e.g. D4
  • muscarinic Ach receptors e.g. Ml
  • nicotinic acetylcholine receptor e.g.
  • neurotransmitter transporters - norepinephrine transporter (NET), dopamine transporter (DAT), serotonin transporter (SERT), vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), glutamate transporter (e.g. GLTl), vesicular acetylcholine transporter, choline transporter (e.g. CHTl); other protein biomarkers include, but not limited to vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2, MMP9.
  • NR1A2B Glutamate receptor subunits
  • AMPA Glutamate receptor subunits
  • Kainate receptors e.g. GIuRl, GluR4
  • beta-adrenoceptor subtypes e.g. beta(2)
  • Alpha- adrenoceptors subtypes e.g. alpha(2c)
  • GABA receptors e.g. GABA(B)
  • Metabotropic glutamate receptor e.g. mGluR3
  • 5-HT serotonin receptors e.g. 5-HT(3)
  • Dopamine receptors e.g. D4
  • Muscarinic Ach receptors e.g. Ml
  • Nicotinic Acetylcholine Receptor e.g.
  • Neurotransmitter Transporters Norepinephrine Transporter (NET), Dopamine transporter (DAT), Serotonin transporter (SERT), Vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLTl), Vesicular acetylcholine transporter, Vesicular Glutamate Transporter 1, [VGLUTl; BNPI] and VGLUT2, Choline transporter, (e.g.
  • CHTl Cholinergic Biomarkers: Acetylcholine Esterase, Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers: Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers: Phenylethanolamine N-methyltransferase (PNMT); Serotonergic Biomarkers: Tryptophan
  • TrH Hydroxylase
  • Glutamatergic Biomarkers Glutaminase, Glutamine synthetase
  • GABAergic Biomarkers GABA transaminase [GABAT]), GABA-B-R2.
  • a patient can be treated with anti-inflammatory agents (e.g. steroidal or non-steroidal) and/or anti-cytokine antibodies to decrease the inflammation.
  • anti-inflammatory agents e.g. steroidal or non-steroidal
  • anti-cytokine antibodies are well know to one of ordinary skill in the art.
  • a composition of markers for brain injury comprise: IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70),
  • IL-13 Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-
  • antibodies specific for cytokines comprise IL-I,
  • IL-l ⁇ IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13,
  • Angiogenin GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -
  • NGF NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ .
  • the sample is selected from the group consisting of saliva, sputum, blood, blood plasma, serum, urine, tissue, cells, and liver.
  • a method of treating a patient suffering from neurological disorders comprising measuring the cytokine levels in the patients and treating the patient with anti-inflammatory agents.
  • a patient is treated with agents that decrease cytokine levels identified as diagnostic of neurological damage as compared to a normal individual.
  • decreasing the cytokine levels decreases neurological injury.
  • a method of monitoring effectiveness of treatment of neural injury comprises measuring cytokine levels and/or total protein in a biological sample obtained from said subject, wherein detected levels of cytokines and/or total protein compared to normal subjects is indicative of the effectiveness of treatment of neurological injury.
  • decreased levels of cytokines and/or total protein in a biological sample obtained from said subject, as compared to levels detected prior to treatment in the same patient, is prognostic of the effectiveness of treatment.
  • a method of identifying the severity of neurological injury course in a patient comprising measuring cytokines and/or total protein in a biological sample obtained from said subject, wherein an elevated level of cytokines and/or total protein compared to normal subjects is indicative of the severity of neurological injury.
  • decreased levels of cytokines and/or total protein in a biological sample obtained from said subject, as compared to levels detected prior to treatment in the same patient, is prognostic of the effectiveness of treatment.
  • the sample is selected from the group consisting of cerebrospinal fluid, blood, blood plasma, serum, urine, tissue, cells, and organs.
  • cytokine levels and/or total protein are detected using an immunoassay.
  • an immunoassay for example, ELISA, RIA and the like.
  • the cytokine levels and/or total protein are detected using a biochip array.
  • the biochip array is a protein chip array.
  • the biochip array is a nucleic acid array.
  • the biochip can comprise one or more antibodies specific for cytokines; single or double stranded nucleic acids; proteins, peptides or fragments thereof; amino acid probes; phage display libraries; one or more cytokines and/or total proteins.
  • kits comprises IL-I, IL-l ⁇ , IL-2, IL-4, IL-5,
  • IL-6 IL-8
  • IL-10 IL-Il
  • IL-12 p40
  • IL-12 p70
  • IL-13 Angiogenin
  • GM-CSF GM-CSF
  • IFN- ⁇ IFN- ⁇
  • MCP-I IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I,
  • Figures IA and IB are graphs showing the results from immunological (ELISA) assays for cytokines IL-6 ( Figure IA) and IL-10 ( Figure IB) in serum showing significant increases after Meth and MDMA acute overdose and traumatic brain injury.
  • ELISA immunological
  • Meth and MDMA showed statistically significant increases in IL-6 at 24 hours after treatment (p values of 0.0043 and 0.0258 respectively).
  • Meth also showed a statistically significant increase in IL-10 at 24 hours after treatment (p value of 0.0003).
  • rat brain trauma 1.6 mm severe control cortical impact
  • Figure 2A are the results from cortex.
  • Figure 2B are the results from hippocampus. These data are indicative of CNS injury and neuronal cell death reflecting a marked similarity between acute Meth injury and mechanical brain injury.
  • Figure 3 is a graph showing cytokine IL-6 ELISA assay of clinical TBI samples.
  • CSF and serum samples were collected from TBI patients at the indicated time points following injury and were compared with non-trauma CSF and serum control samples (n values as indicated).
  • Figure 4 is a graph showing changes in rat weight after acute METH administration and TBI insult.
  • saline treatments white bars
  • n 4.
  • Rat weight decreases significantly following the 24 hrs drug administration which reached approximately 12% of the total body weight. This weight loss was not significant in TBI rats.
  • Results are expressed as mean ⁇ SEM gms and * marks above bars represent the statistical significance between the groups at the level of P ⁇ 0.05. Student's t tests was applied for statistical significance (p ⁇ 0.005 for each).
  • Figures 5A-5B show the serum analysis of the rat cytokine antibody array. Serum analysis of the rat cytokine antibody array revealed a correlation between increasing levels of certain cytokines with the two brain insults (acute METH administration and TBI). A panel of 19 secreted cytokines was checked for any changes in the serum of control saline rats, acute METH administration and TBI insults by using the RayBiotec cytokine array kit.
  • Figure 5A A representative cytokine blot is shown from the saline control, METH treated and TBI samples.
  • the boxes bordering differential spots on the blots demonstrate the cytokines that are up-regulated which included IL- l ⁇ , IL-6 and IL-10 compared with the saline control serum.
  • Figure 5B The actual cytokine array map from RayBioTM is also provided; it detects GM-CSF, IFN- ⁇ , IL-l- ⁇ , IL-4, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MIP- 3 ⁇ , MCP-I, LIX, IL-10, TNF- ⁇ , VEGF and IL-6.
  • Figures 6A-6B are graphs showing altered cytokines analysis in the rat serum after acute meth administration and TBI Insult. Graphical representation of the up-regulated cytokines following the acute METH treatment (grey bars) compared to saline control (white bars), significant changes were observed (IL- l ⁇ , IL-6 and IL-IO cytokines) shown in Figure 6A. In Figure 6B, graphical representation of the up-regulated cytokines following the TBI insult (dashed bars) compared to saline control, significant changes were observed (IL- l ⁇ , IL- 6 and IL-10 cytokines).
  • FIGS. 7A-7B are graphs showing ELISA quantitation of serum IL-6 and IL-10 cytokines after acute METH administration and TBI insult. Serum concentrations of respective IL-6 and IL-10 after saline treatment (white bars) acute METH treatment (grey bars) and TBI rats (dashed bars). IL-6 and IL-10 levels were increased significantly in similar manners after acute METH treatments and TBI insult. Results are expressed as mean +/- SEM pg/ml and * marks above bars represent the statistical significance between the groups at the level of P ⁇ 0.05. Student's t-tests was applied for statistical significance (p ⁇ 0.005 for each).
  • the present invention provides methods of diagnostics and treatment of acute chemical brain injury including drug abuse brain injury due to acute exposure to methamphetamine (e.g., overdose).
  • the methods are utilized for any form of neurological injury (e.g., chemical injury or mechanical).
  • the term "injury or neural injury” is intended to include a damage which directly or indirectly affects the normal functioning of the CNS and PNS.
  • the injury can be damage to retinal ganglion cells; a traumatic brain injury; a stroke related injury; a cerebral aneurism related injury; a spinal cord injury, including monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia; a neuroproliferative disorder or neuropathic pain syndrome.
  • Neurological or neural injury includes damage to the brain due to drugs.
  • neurological injuries include: acute spinal cord trauma, spinal cord compression, spinal cord hematoma, cord contusion (these cases are usually traumatic, such as motorcycle accidents or sports injuries); nerve compression, the most common condition being a herniated disc causing sciatic nerve compression, neuropathy, and pain; but also including cervical disc herniation, causing nerve compression in the neck; carpal tunnel syndrome (non-RA); acute or chronic spinal cord compression from cancer (this is usually due to metastases to the spine, such as from prostate, breast or lung cancer); autoimmune disease of the nervous system; and demyelinating diseases, the most common condition being multiple sclerosis.
  • CBI chemical brain injury
  • CNS injuries or disease include TBI, stroke, concussion (including post-concussion syndrome), cerebral ischemia, neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease and Alzheimer's disease, Creutzfeldt-Jakob disease, brain injuries secondary to seizures which are induced by radiation, exposure to ionizing or iron plasma, nerve agents, cyanide, toxic concentrations of oxygen, neurotoxicity due to CNS malaria or treatment with anti-malaria agents, trypanosomes, malarial pathogens, and other CNS traumas.
  • TBI TBI
  • stroke concussion (including post-concussion syndrome)
  • cerebral ischemia neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease and Alzheimer's disease, Creutzfeldt-Jakob disease
  • brain injuries secondary to seizures which are induced by radiation, exposure to ionizing or iron plasma
  • nerve agents nerve agents
  • cyanide
  • the term "Traumatic Brain Injury” is art recognized and is intended to include the condition in which, a traumatic blow to the head causes damage to the brain, often without penetrating the skull.
  • the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF).
  • ICP intracranial pressure
  • CBF cerebral blood flow
  • cytokine is used generally.
  • the term includes: lymphokine (cytokines produced by lymphocytes), monokine (cytokines produced by monocytes), chemokine (cytokines with chemotactic activities), and interleukin (cytokines produced by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action).
  • Pro-inflammatory cytokines are cytokines produced predominantly by activated immune cells such as microglia and are involved in the amplification of inflammatory reactions. These include IL-I, IL-6, TNF- ⁇ , and TGF- ⁇ .
  • Anti-inflammatory cytokines belong to the T cell-derived cytokines and are involved in the down -regulation of inflammatory reactions.
  • the anti-inflammatory cytokines are a series of immunoregulatory molecules that control the proinflammatory cytokine response. Cytokines act in concert with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response.
  • Major anti-inflammatory cytokines include interleukin (IL)-I receptor antagonist, IL-4, IL-6, IL-IO, IL-Il, and IL-13.
  • IL-I receptor antagonists for IL-I, tumor necrosis factor-alpha, and IL- 18 also function as proinflammatory cytokine inhibitors.
  • a marker can be a polypeptide which is present at an elevated level or at a decreased level in samples of patients with neural injury compared to samples of control subjects.
  • a marker can be a polypeptide which is detected at a higher frequency or at a lower frequency in samples of patients compared to samples of control subjects.
  • a marker can be differentially present in terms of quantity, frequency or both.
  • a polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is statistically significantly different from the amount of the polypeptide in the other sample.
  • a polypeptide is differentially present between the two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other.
  • a polypeptide is differentially present between the two sets of samples if the frequency of detecting the polypeptide in samples of patients' suffering from neural injury and/or neuronal disorders, is statistically significantly higher or lower than in the control samples.
  • a polypeptide is differentially present between the two sets of samples if it is detected at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% more frequently or less frequently observed in one set of samples than the other set of samples.
  • "Diagnostic" means identifying the presence or nature of a pathologic condition.
  • Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
  • the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • test amount of a marker refers to an amount of a marker present in a sample being tested.
  • a test amount can be either in absolute amount (e.g., ⁇ g/ml) or a relative amount (e.g., relative intensity of signals).
  • a "diagnostic amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of neural injury and/or neuronal disorder.
  • a diagnostic amount can be either in absolute amount (e.g., ⁇ g/ml) or a relative amount (e.g., relative intensity of signals).
  • a "control amount" of a marker can be any amount or a range of amount which is to be compared against a test amount of a marker.
  • a control amount of a marker can be the amount of a marker in a person without neural injury and/or neuronal disorder.
  • a control amount can be either in absolute amount (e.g., ⁇ g/ml) or a relative amount (e.g., relative intensity of signals).
  • sample is used herein in its broadest sense.
  • a sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
  • Neurodegenerative disorders Parkinson's; Alzheimer's) or autoimmune disorders (multiple sclerosis) of the central nervous system; memory loss; long term and short term memory disorders; learning disorders; autism, depression, benign forgetfulness, childhood learning disorders, close head injury, and attention deficit disorder; autoimmune disorders of the brain, neuronal reaction to viral infection; brain damage; depression; psychiatric disorders such as bi-polarism, schizophrenia and the like; narcolepsy/sleep disorders(including circadian rhythm disorders, insomnia and narcolepsy); severance of nerves or nerve damage; severance of the cerebrospinal nerve cord (CNS) and any damage to brain or nerve cells; neurological deficits associated with AIDS; tics (e.g.
  • Giles de Ia Tourette's syndrome Huntington's chorea, schizophrenia, traumatic brain injury, tinnitus, neuralgia, especially trigeminal neuralgia, neuropathic pain, inappropriate neuronal activity resulting in neurodysthesias in diseases such as diabetes, MS and motor neurone disease, ataxias, muscular rigidity (spasticity) and temporomandibular joint dysfunction; Reward Deficiency Syndrome (RDS) behaviors in a subject.
  • RDS Reward Deficiency Syndrome
  • a composition of biomarkers comprises IL-I, IL- l ⁇ ,
  • IL-2 IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM-
  • CSF IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2,
  • antibodies specific for biomarkers of neurological injury comprise antibodies specific for IL-I, IL- l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8,
  • IL-10 IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10,
  • MIP-Ib MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ .
  • a method of diagnosing neurological injury in a patient comprises identifying cytokines and cytokine levels in a sample from an injured patient, wherein the cytokines detected are elevated as compared to normal individuals.
  • detected cytokines include, but not limited to: IL-I, IL- l ⁇ , IL-2, IL-4, IL-5, IL-
  • IL-8 IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I,
  • the sample is selected from the group consisting of saliva, sputum, blood, blood plasma, serum, urine, tissue, cells, and liver.
  • the cytokine levels are compared to known biomarkers of neurological injury. For example, ⁇ II spectrin, spectrin break down products,
  • a method of treating a patient suffering from neurological disorders comprises measuring the cytokine levels in the patients and treating the patient with compounds that decrease the cytokine levels to levels indicative of a normal individual.
  • the patient is treated with anti-inflammatory agents and/or agents that decrease inflammatory cytokine production.
  • treatment of a patient comprises administering one or more anti-inflammatory agent, anti-inflammatory cytokines, cytokine inhibitors such as for example, receptor blockers.
  • a method of monitoring effectiveness of treatment of neural injury comprises measuring cytokine levels and/or total protein in a biological sample obtained from said subject, wherein detected levels of cytokines and/or total protein compared to normal subjects is indicative of the effectiveness of treatment of neurological injury.
  • a method of identifying the severity of neurological injury course in a patient comprises measuring cytokines and/or total protein in a biological sample obtained from said subject, wherein an elevated level of cytokines and/or total protein compared to normal subjects is indicative of the severity of neurological injury.
  • treatment of a patient suffering from neurological injury comprises administration of one or more antibodies to IL-I, IL- l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ protein or fragments thereof.
  • the treatment can be in conjunction with administration of one or more ant-inflammatory agents which include both steroidal and non-steroidal agents, analgesics and the like.
  • the cytokine biomarkers of neurological injury are correlated to other biomarkers of neurological injury.
  • neural proteins such as for example, axonal proteins - NF-200 (NF-H), NF- 160 (NF-M), NF- 68 (NF-L); amyloid precursor protein; dendritic proteins - alpha-tubulin (P02551), beta- tubulin (PO 4691), MAP-2A/B, MAP-2C, Tau, Dynamin-1 (P21575), Dynactin (Q13561), P24; somal proteins - UCH-Ll (Q00981), PEBP (P31044), NSE (P07323), Thy 1.1, Prion, Huntington; presynaptic proteins - synapsin-1, synapsin-2, alpha- synuclein (p37377), beta- synuclein (Q63754), GAP43, synaptophysin,
  • neural proteins such as for example, axonal proteins
  • alpha-adrenoreceptor subtypes (e.g. (alpha (2c)), GABA receptors (e.g. GABA(B)), metabotropic glutamate receptor (e.g. mGluR3), NMDA receptor subunits (e.g. NR1A2B), Glutamate receptor subunits (e.g. GluR4), 5-HT serotonin receptors (e.g. 5-HT(3)), dopamine receptors (e.g. D4), muscarinic Ach receptors (e.g. Ml), nicotinic acetylcholine receptor (e.g.
  • neurotransmitter transporters - norepinephrine transporter (NET), dopamine transporter (DAT), serotonin transporter (SERT), vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), glutamate transporter (e.g. GLTl), vesicular acetylcholine transporter, choline transporter (e.g. CHTl); other protein biomarkers include, but not limited to vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2, MMP9.
  • NR1A2B Glutamate receptor subunits
  • AMPA Glutamate receptor subunits
  • Kainate receptors e.g. GIuRl, GluR4
  • beta-adrenoceptor subtypes e.g. beta(2)
  • Alpha- adrenoceptors subtypes e.g. alpha(2c)
  • GABA receptors e.g. GABA(B)
  • Metabotropic glutamate receptor e.g. mGluR3
  • 5-HT serotonin receptors e.g. 5-HT(3)
  • Dopamine receptors e.g. D4
  • Muscarinic Ach receptors e.g. Ml
  • Nicotinic Acetylcholine Receptor e.g.
  • Neurotransmitter Transporters Norepinephrine Transporter (NET), Dopamine transporter (DAT), Serotonin transporter (SERT), Vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLTl), Vesicular acetylcholine transporter, Vesicular Glutamate Transporter 1, [VGLUTl; BNPI] and VGLUT2, Choline transporter, (e.g.
  • CHTl Cholinergic Biomarkers: Acetylcholine Esterase, Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers: Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers: Phenylethanolamine N-methyltransferase (PNMT); Serotonergic Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers: Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA transaminase [GABAT]), GABA-B-R2.
  • the amount of cytokine marker detected is diagnostic of the extent of damage or injury. Quantitation of each biomarker is described in the specification and in the Examples to follow. Assays include immunoassays (such as ELISA' s), spectrophotometry, HPLC, SELDI, biochips and the like. The quantitation of each as compared to a normal individual is diagnostic of the extent of injury.
  • neurological damage in a subject is analyzed by (a) providing a biological sample isolated from a subject suspected of having a neurological injury; (b) detecting in the sample the presence or amount of at least one marker selected from one or more cytokines; and (c) correlating the presence or amount of the cytokines with the presence or type of neurological injury in the subject.
  • the nervous system such as brain injury
  • the neural cell membrane is compromised, leading to the efflux of these neural proteins first into the extracellular fluid or space and to the cerebrospinal fluid and eventually in the circulating blood (as assisted by the compromised blood brain barrier) and other biofluids (e.g. urine, sweat, s saliva, etc.).
  • suitable biological samples include, but not limited to such cells or fluid secreted from these cells.
  • Obtaining biological fluids such as cerebrospinal fluid, blood, plasma, serum, saliva and urine, from a subject is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample.
  • samples, which are biological fluids are preferred for use in the invention.
  • CSF in particular, is preferred for detecting nerve damage in a subject as it is in immediate contact with the nervous system and is readily obtainable.
  • a biological sample can be obtained from a subject by conventional techniques. For example, CSF can be obtained by lumbar puncture. Blood can be obtained by venipuncture, while plasma and serum can be obtained by fractionating whole blood according to known methods.
  • Surgical techniques for obtaining solid tissue samples are well known in the art.
  • methods for obtaining a nervous system tissue sample are described in standard neuro-surgery texts such as Atlas of Neurosurgery: Basic Approaches to Cranial and Vascular Procedures, by F. Meyer, Churchill Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain Tumors, 1st ed., by David G.T. Thomas, WB Saunders Co., 1993; and Cranial Microsurgery: Approaches and Techniques, by L. N. Sekhar and E. De Oliveira, 1st ed., Thieme Medical Publishing, 1999.
  • Methods for obtaining and analyzing brain tissue are also described in Belay et al., Arch. Neurol. 58: 1673-1678 (2001); and Seijo et al., J. Clin. Microbiol. 38: 3892-3895 (2000).
  • any animal that expresses neural proteins can be used as a subject from which a biological sample is obtained.
  • the subject is a mammal, such as for example, a human, dog, cat, horse, cow, pig, sheep, goat, primate, rat, mouse and other vertebrates such as fish, birds and reptiles. More preferably, the subject is a human.
  • Particularly preferred are subjects suspected of having or at risk for developing traumatic or non-traumatic nervous system injuries, such as victims of brain injury caused by traumatic insults (e.g. gunshots wounds, automobile accidents, sports accidents, shaken baby syndrome), ischemic events (e.g.
  • neurodegenerative disorders such as Alzheimer's, Huntington's, and Parkinson's diseases; Prion-related disease; other forms of dementia, and spinal cord degeneration
  • epilepsy substance abuse (e.g., from amphetamines, methamphetamine/Speed , Ecstasy/MDMA, or ethanol and cocaine), and peripheral nervous system pathologies such as diabetic neuropathy, chemotherapy-induced neuropathy and neuropathic pain, peripheral nerve damage or atrophy (ALS), multiple sclerosis (MS).
  • substance abuse e.g., from amphetamines, methamphetamine/Speed , Ecstasy/MDMA, or ethanol and cocaine
  • peripheral nervous system pathologies such as diabetic neuropathy, chemotherapy-induced neuropathy and neuropathic pain, peripheral nerve damage or atrophy (ALS), multiple sclerosis (MS).
  • ALS peripheral nerve damage or atrophy
  • MS multiple sclerosis
  • the invention provides the step of correlating the presence or amount of one or more cytokines and/or total protein with the severity and/or type of neural injury.
  • the amount of a cytokines, total protein, neural proteins, peptides, fragments, derivatives or the modified forms, thereof directly relates to severity of nerve tissue injury as more severe injury damages a greater number of nerve cells which in turn causes a larger amount of neural protein(s) to accumulate in the biological sample (e.g., serum, plasma, blood, CSF).
  • Cytokines and Inflammation The concept that the brain is an immunologically privileged organ has been challenged by recent evidence that inflammatory-like processes and immune reactions occur in the CNS after a large variety of peripheral or local stimuli. Cytokines are expressed at very low levels in healthy brain tissue under physiologic conditions; however, they can be rapidly induced there after a variety of injuries. In particular, production of proinflammatory cytokines is an early cellular event subsequent to ischemic, traumatic, and excito toxic insults.
  • Proinflammatory cytokines such as interleukin (IL)-l ⁇ , IL-6, IL-10 and tumor necrosis factor (TNF)- ⁇ , produce detrimental effects on brain function. The effects are dependent on a number of factors, including local concentration of the cytokine at the site of synthesis, the type of target cells, the length of time the tissue is exposed to cytokines, and which receptor subtypes are involved.
  • Microglia and astrocytes are the first cells to produce cytokines during epileptic activity, and they represent the main source of local cytokine production in brain.
  • An additional source of production consists of blood monocytes, pervading the brain several hours after acute epileptic events.
  • Cytokines make up the fourth major class of soluble intercellular signaling molecules, alongside neurotransmitters, endocrine hormones, and autacoids. They possess typical hormonal activities: they are secreted by a single cell type, react specifically with other cell types (target cells) and regulate specific vital functions that are controlled by feedback mechanisms; they generally act at short range in a paracrine or autocrine (rather than endocrine) manner; they interact first with high-affinity cell surface receptors (distinct for each type or even subtype) and then regulate the transcription of a number of cellular genes by little understood second signals. This altered transcription (which can be an enhancement or inhibition) result in changes in cell behavior.
  • Target cells on which cytokines transform their information signal, may be localized in any body compartment (sometimes a long distance from the site of secretion). Other type of these molecules act mostly on neighboring cells in the microenvironment where they have been released. These are characterized as local hormones and their secretion is brought about by autocrine (only the cell or organ of secretion is affected) or paracrine mechanisms. During the paracrine secretion some cytokines may escape cell binding and may spill over into general circulation via lymph or plasma.
  • lymphoid cells which are mobile after having picked up the message in the microenvironment throughout the body and therefore their immunoregulatory products, (lymphokines, monokines, interleukins and other cytokines), despite being of local hormone character, may act in fact systemically.
  • cytokines The central role of cytokines is to control the direction, amplitude, and duration of immune responses and to control the (re)modeling of tissues, be it developmentally programmed, constitutive, or unscheduled. Unscheduled remodeling is that which accompanies inflammation, infection, wounding, and repair. Individual cytokines can have pleiotropic (multiple), overlapping and sometimes contradictory functions depending on their concentration, the cell type they are acting on, and the presence of other cytokines and mediators. Thus the information which an individual cytokine conveys depend on the pattern of regulators to which a cell is exposed, and not on one single cytokine. It is supposed that all cytokines form the specific system or network of communication signals between cells of the immune system, and between the immune system and other organs. In this inter-cell signaling network, the signal is usually transferred by means of a special set of cytokines.
  • Th2 lymphocyte development T cells (Th2), mast cells, B Promotes Th2 lymphocyte development; inhibition of
  • IL-4 cells IL-4 cells
  • stromal cells LPS-induced proinflammatory cytokine synthesis
  • T cells B cells, monocytes, Inhibition of TNF and IL-I production by
  • IL-IO cells (Th2)
  • B cells lymphocyte responses
  • TGF- ⁇ many cell lines expression and proinflammatory cytokine synthesis
  • IL- 18 binding protein Splenocytes multiple that functions as a decoy receptor and binds (IL- 18BP) other cell lines circulating IL- 18 Detection of Cytokine Biomarkers
  • biomarkers of the invention can be detected in a sample by any means. Methods for detecting the biomarkers are described in detail in the materials and methods and Examples which follow.
  • immunoassays include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, fluorescent immunoassays and the like.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding an antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4°C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4°C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxy
  • the ability of the antibody to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or n T) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the anti
  • ELISAs comprise preparing antigen (i.e.
  • the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
  • biochips are protein biochips for capture and detection of proteins e.g. cytokines, antibodies etc.
  • proteins e.g. cytokines, antibodies etc.
  • Many protein biochips are described in the art. These include, for example, protein biochips produced by Packard BioScience Company (Meriden CT), Zyomyx (Hayward, CA) and Phylos (Lexington, MA).
  • protein biochips comprise a substrate having a surface. A capture reagent or adsorbent is attached to the surface of the substrate. Frequently, the surface comprises a plurality of addressable locations, each of which location has the capture reagent bound there.
  • the capture reagent can be a biological molecule, such as a polypeptide or a nucleic acid, which captures other biomarkers in a specific manner.
  • the capture reagent can be a chromatographic material, such as an anion exchange material or a hydrophilic material. Examples of such protein biochips are described in the following patents or patent applications: U.S.
  • a sample containing the biomarkers or antibodies to a biomarker e.g. IL-I, IL-6, IL-10
  • a biomarker e.g. IL-I, IL-6, IL-10
  • a suitable eluant e.g. IL-I, IL-6, IL-10
  • Analytes captured on the surface of a protein biochip can be detected by any method known in the art. This includes, for example, mass spectrometry, fluorescence, surface plasmon resonance, ellipsometry and atomic force microscopy. Mass spectrometry, and particularly SELDI mass spectrometry, is a particularly useful method for detection of the biomarkers of this invention.
  • a laser desorption time-of-flight mass spectrometer is used in embodiments of the invention.
  • a substrate or a probe comprising markers is introduced into an inlet system.
  • the markers are desorbed and ionized into the gas phase by laser from the ionization source.
  • the ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of-flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of markers of specific mass to charge ratio.
  • MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry
  • MALDI-MS is a method of mass spectrometry that involves the use of an energy absorbing molecule, frequently called a matrix, for desorbing proteins intact from a probe surface.
  • MALDI is described, for example, in U.S. patent 5,118,937 (Hillenkamp et al.) and U.S. patent 5,045,694 (Beavis and Chait).
  • the sample is typically mixed with a matrix material and placed on the surface of an inert probe.
  • Exemplary energy absorbing molecules include cinnamic acid derivatives, sinapinic acid (“SPA”), cyano hydroxy cinnamic acid (“CHCA”) and dihydroxybenzoic acid. Other suitable energy absorbing molecules are known to those skilled in this art.
  • the matrix dries, forming crystals that encapsulate the analyte molecules. Then the analyte molecules are detected by laser desorption/ionization mass spectrometry.
  • MALDI-MS is useful for detecting the biomarkers of this invention if the complexity of a sample has been substantially reduced using the preparation methods described above.
  • SELDI-MS Surface-enhanced laser desorption/ionization mass spectrometry, or SELDI-MS represents an improvement over MALDI for the fractionation and detection of biomolecules, such as proteins, in complex mixtures.
  • SELDI is a method of mass spectrometry in which biomolecules, such as proteins, are captured on the surface of a protein biochip using capture reagents that are bound there. Typically, non-bound molecules are washed from the probe surface before interrogation.
  • SELDI is described, for example, in: United States Patent 5,719,060 ("Method and Apparatus for Desorption and Ionization of Analytes," Hutchens and Yip, February 17, 1998,) United States Patent 6,225,047 ("Use of Retentate Chromatography to Generate Difference Maps," Hutchens and Yip, May 1, 2001) and Weinberger et al., "Time-of-flight mass spectrometry," in Encyclopedia of Analytical Chemistry, R.A. Meyers, ed., pp 11915-11918 John Wiley & Sons Chichesher, 2000.
  • Markers on the substrate surface can be desorbed and ionized using gas phase ion spectrometry.
  • Any suitable gas phase ion spectrometers can be used as long as it allows markers on the substrate to be resolved.
  • gas phase ion spectrometers allow quantitation of markers.
  • a gas phase ion spectrometer is a mass spectrometer.
  • a substrate or a probe comprising markers on its surface is introduced into an inlet system of the mass spectrometer.
  • the markers are then desorbed by a desorption source such as a laser, fast atom bombardment, high energy plasma, electrospray ionization, thermospray ionization, liquid secondary ion MS, field desorption, etc.
  • the generated desorbed, volatilized species consist of preformed ions or neutrals which are ionized as a direct consequence of the desorption event.
  • Generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions.
  • the ions exiting the mass analyzer are detected by a detector.
  • the detector then translates information of the detected ions into mass-to-charge ratios. Detection of the presence of markers or other substances will typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of markers bound to the substrate.
  • Any of the components of a mass spectrometer e.g., a desorption source, a mass analyzer, a detector, etc.
  • an immunoassay can be used to detect and analyze markers in a sample. This method comprises: (a) providing an antibody that specifically binds to a marker; (b) contacting a sample with the antibody; and (c) detecting the presence of a complex of the antibody bound to the marker in the sample.
  • nucleic acid and amino acid sequences for markers can be obtained by further characterization of these markers.
  • each marker can be peptide mapped with a number of enzymes (e.g., trypsin, V8 protease, etc.).
  • the molecular weights of digestion fragments from each marker can be used to search the databases, such as SwissProt database, for sequences that will match the molecular weights of digestion fragments generated by various enzymes.
  • the nucleic acid and amino acid sequences of other markers can be identified if these markers are known proteins in the databases.
  • the proteins can be sequenced using protein ladder sequencing.
  • Protein ladders can be generated by, for example, fragmenting the molecules and subjecting fragments to enzymatic digestion or other methods that sequentially remove a single amino acid from the end of the fragment. Methods of preparing protein ladders are described, for example, in International Publication WO 93/24834 (Chait et al.) and United States Patent 5,792,664 (Chait et al.). The ladder is then analyzed by mass spectrometry. The difference in the masses of the ladder fragments identify the amino acid removed from the end of the molecule.
  • Any suitable method can be used to detect a marker or markers in a sample.
  • an immunoassay or gas phase ion spectrometry can be used as described above. Using these methods, one or more markers can be detected.
  • a sample is tested for the presence of a plurality of markers. Detecting the presence of a plurality of markers, rather than a single marker alone, would provide more information for the diagnostician. Specifically, the detection of a plurality of markers in a sample would increase the percentage of true positive and true negative diagnoses and would decrease the percentage of false positive or false negative diagnoses.
  • the detection of the marker or markers is then correlated with a probable diagnosis of neural injury and/or neuronal disorders.
  • the detection of the mere presence or absence of a marker, without quantifying the amount of marker is useful and can be correlated with a probable diagnosis of neural injury and/or neuronal disorders.
  • the detection of markers can involve quantifying the markers to correlate the detection of markers with a probable diagnosis of neural injury, degree of severity of neural injury, diagnosis of neural disorders and the like. Thus, if the amount of the markers detected in a subject being tested is higher compared to a control amount, then the subject being tested has a higher probability of having such injuries and/or neural disorders.
  • the detection of markers can further involve quantifying the markers and/or total protein to correlate the detection of markers with a probable diagnosis of neural injury, degree of severity of neural injury, diagnosis of neural disorders and the like, wherein the markers are present in lower quantities in CSF or blood serum samples from patients than in blood serum samples of normal subjects.
  • the amount of the markers detected in a subject being tested is lower compared to a control amount, then the subject being tested has a higher probability of having neural injury and/or neural disorder.
  • a control can be, e.g. , the average or median amount of marker present in comparable samples of normal subjects in whom neural injury and/or neural disorders, is undetectable.
  • the control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount. For example, if a test sample is obtained from a subject's cerebrospinal fluid and/or blood serum sample and a marker is detected using a particular probe, then a control amount of the marker is preferably determined from a serum sample of a patient using the same probe. It is preferred that the control amount of marker is determined based upon a significant number of samples from normal subjects who do not have neural injury and/or neuronal disorders so that it reflects variations of the marker amounts in that population.
  • Data generated by mass spectrometry can then be analyzed by a computer software.
  • the software can comprise code that converts signal from the mass spectrometer into computer readable form.
  • the software also can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a "peak" in the signal corresponding to a marker of this invention, or other useful markers.
  • the software also can include code that executes an algorithm that compares signal from a test sample to a typical signal characteristic of "normal" and human neural injury and determines the closeness of fit between the two signals.
  • the software also can include code indicating which the test sample is closest to, thereby providing a probable diagnosis.
  • Antibodies directed against any one of the cytokine biomarkers e.g., IL-I, IL- l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM- CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ , fragments, variants and mutants thereof) can be used, as taught by the present invention, to detect and diagnose neurological injury.
  • Various histological staining methods including immunohistochemical staining methods, may also be used effectively according to the teaching of the invention.
  • One screening method for determining whether a sample contains, for example, IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ - NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ proteins, peptides or fragments thereof comprises, for example, immunoassays employing radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA) methodologies, based on the production of specific antibodies (monoclonal or polyclonal) to IL-10 protein.
  • RIA radioimmunoassay
  • ELISA enzyme- linked immunosorbent assay
  • any sample can be used, however, preferred samples comprising the biomarkers are blood, serum, plasma. Venipuncture (blood), urine and other body secretions, such as sweat and tears, can also be used as biological samples.
  • RIA the substance under test is mixed with diluted antiserum in the presence of radiolabeled antigen. In this method, the concentration of the test substance is inversely proportional to the amount of labeled antigen bound to the specific antibody and directly related to the amount of free labeled antigen.
  • Other suitable screening methods is readily apparent to those of skill in the art.
  • the present invention also relates to methods of detecting cytokine biomarker proteins or fragments thereof, in a sample or subject.
  • cytokine biomarker proteins or fragments thereof may be detectably labeled with any appropriate marker, for example, a radioisotope, an enzyme, a fluorescent label, a paramagnetic label, or a free radical.
  • antibody refers both to monoclonal antibodies, which are a substantially homogeneous population and to polyclonal antibodies, which are heterogeneous populations.
  • Polyclonal antibodies are derived from the sera of animals immunized with an antigen.
  • Monoclonal antibodies (MAbs) to specific antigens may be obtained by methods known to those skilled in the art. See, for example, U.S. Pat. No. 4,376,110.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • Fab and F(ab') 2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of an for example, IL-10 proteins, peptides or fragments thereof, according to the methods disclosed herein in order to detect and diagnose neurological injury or associated diseases thereof in the same manner as an intact antibody.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitopic determinants are meant to refer to that portion of any molecule capable of being bound by an antibody that can also be recognized by that antibody.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • an "antigen" is a molecule capable of being bound by an antibody that is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen may have one, or more than one epitope.
  • the specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies that may be evoked by other antigens.
  • the antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the cytokine biomarkers.
  • the antibodies (or fragments thereof) useful in the present invention may be employed histologically to detect or visualize the presence of IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ , proteins, peptides, or fragments thereof.
  • Such an assay for detecting biomarkers typically comprises incubating a biological sample from a subject suspected of having such a condition in the presence of a detectably labeled binding molecule (e.g., antibody) capable of identifying a biomarker and detecting the binding molecule which is bound in a sample.
  • a biological sample may be treated with nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled, with for example, anti-IL-l ⁇ , and/or anti-IL-6 and/or anti-IL-10 specific antibody.
  • solid phase support any support capable of binding antigen or antibodies.
  • supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads.
  • One embodiment for carrying out the diagnostic assay of the present invention on a biological sample containing cytokine biomarkers comprises contacting a detectably labeled antibody specific for a desired biomarker.
  • 11-10 is used as a non-limiting example.
  • a detectably labeled anti- IL-10 specific antibody is bound to a solid support to effect immobilization of anti-IL-10 specific antibody; contacting a sample suspected of containing IL-10 or fragments thereof on the said solid support; incubating the detectably labeled anti-IL-10 specific antibody with the support for a time sufficient to allow the immobilized anti-IL-10 specific antibody to bind to IL-10 and fragments thereof. These steps are followed by washing and detecting the bound label and thereby detecting and quantifying IL-10 and fragments thereof.
  • labeled anti-IL-10 specific antibody and/or IL-10 protein complexes in a sample may be separated from a reaction mixture by contacting the complex with an immobilized antibody or protein which is specific for an immunoglobulin, e.g., Staphylococcus protein A, Staphylococcus protein G, anti-IgM or anti-IgG antibodies.
  • an immunoglobulin e.g., Staphylococcus protein A, Staphylococcus protein G, anti-IgM or anti-IgG antibodies.
  • antiimmunoglobulin antibodies may be polyclonal or preferably monoclonal.
  • the solid support may then be washed with a suitable buffer to give an immobilized IL-10/labeled anti- IL-IO specific antibody complex.
  • the label may then be detected to give a measure of IL-10 protein.
  • the specific concentrations of detectably labeled antibody and IL-10, the temperature and time of incubation, as well as other assay conditions may be varied, depending on various factors including the concentration of protein in the sample, the nature of the sample, and the like.
  • the binding activity of a given lot of anti-IL-10 antibody may be determined according to well-known methods. Those skilled in the art is able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation. [0101]
  • One of the ways in which the anti-IL-10 specific antibody can be detectably labeled is by linking the same to an enzyme.
  • Enzymes which can be used to detectably label the anti-IL-10 specific antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, ⁇ -V-steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, ⁇ - galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling the anti-IL-10 specific antibodies or antibody fragments, it is possible to detect IL-10 protein or fragments thereof, through the use of radioimmunoassays.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes that are particularly useful for the purpose of the present invention are: 3 H, 125 I, 131 1, 35 S, 14 C, and preferably 125 I.
  • the anti-IL-10 specific antibody can also be detectably labeled using fluorescence emitting metals such as ] 2 Eu, or others of the lanthanide series. These metals can be attached to the anti-IL-10 specific antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the anti-IL-10 specific antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • chemiluminescent-tagged anti-IL-10 specific antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • the anti-IL-10 specific antibody may also be labeled with biotin and then reacted with avidin.
  • a bioluminescent compound may be used to label the anti-IL-10 specific antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Detection of the anti-IL-10 specific antibody may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material.
  • the detection can be accomplished by calorimetric methods that employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • the IL-10 protein and/or fragments thereof, that are detected by this assay may be present in a biological sample. Any sample containing an IL-10 protein or fragments thereof, can be used.
  • the sample is a biological solution such as, for example, plasma, amniotic fluid, blood, serum, urine and the like.
  • the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions that allow the use of other samples.
  • the diagnosis of neurological injury and/or disease can be established by a simple, non-invasive blood immunoassay that reveals IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ protein levels and/or fragments thereof, greatly increased over normal levels.
  • Radionuclide for in vivo diagnosis is that the half-life of a radionuclide be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host is minimized.
  • a radionuclide used for in vivo imaging will lack a particulate emission, but produce a large number of photons in the 140-200 keV range, which maybe readily detected by conventional gamma cameras.
  • radionuclides may be bound to antibody either directly or indirectly by using an intermediary functional group.
  • Intermediary functional groups that are often used in binding radioisotopes that exist as metallic ions to immunoglobulins are DTPA and EDTA.
  • Typical examples of ions that can be bound to immunoglobulins are 99m Tc, 123 I,
  • the type of detection instrument available is a major factor in selecting a given radionuclide.
  • the radionuclide chosen must have a type of decay that is detectable for a given type of instrument.
  • any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention.
  • PET, gamma, beta, and MRI detectors can be used to visualize diagnostic imagining.
  • the antibodies useful in the invention can also be labeled with paramagnetic isotopes for purposes of in vivo diagnosis. Elements that are particularly useful, as in
  • Magnetic Resonance Imaging include 157 Gd, 55 Mn, 162 Dy, and 56 Fe.
  • the antibodies useful in the present invention are also particularly suited for use in in vitro immunoassays to detect the presence of an IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-
  • MIP-Ib MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and
  • TGF- ⁇ protein or fragments thereof in body tissue, fluids (such as CSF, blood, plasma or serum), or cellular extracts.
  • the antibodies may be utilized in liquid phase or, preferably, bound to a solid-phase carrier, as described above.
  • a solid-phase carrier as described above.
  • Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m- maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.
  • Removing a histological specimen from a patient, and providing the combination of labeled antibodies of the present invention to such a specimen may accomplish in situ detection.
  • the antibody is preferably provided by applying or by overlaying the labeled antibody to a biological sample.
  • the binding molecules of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
  • an immunometric assay also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested (i.e., blood, plasma or serum) and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • Typical, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody- antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.
  • This type of forward sandwich assay may be a simple "yes/no" assay to determine whether antigen is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of antigen.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • Such detection methods may be used to assist in the determination of the stage of neural deterioration in neurological injury and/or disease by evaluating and comparing the concentration of an IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ protein or fragments thereof, in the biological sample.
  • kits for aiding a diagnosis of neural injury wherein the kits can be used to detect the markers of the present invention.
  • the kits can be used to detect any one or more of the markers described herein, which markers are differentially present in samples of a patient and normal subjects.
  • the kits of the invention have many applications.
  • the kits can be used to identify compounds that modulate expression of one or more of the markers in in vitro or in vivo animal models to determine the effects of treatment.
  • kits comprises (a) an antibody that specifically binds to a marker; and (b) a detection reagent.
  • a kit can be prepared from the materials described above, and the previous discussion regarding the materials (e.g., antibodies, detection reagents, immobilized supports, etc.) is fully applicable to this section and will not be repeated.
  • the kit may further comprise pre-fractionation spin columns.
  • the kit may further comprise instructions for suitable operation parameters in the form of a label or a separate insert.
  • the kit comprises (a) a panel or composition of biomarkers (b) a detecting agent.
  • the panel or composition of biomarkers included in a kit include at least one biomarker and/or a plurality of biomarkers in order to diagnose in vivo location of neural injury.
  • biomarkers include: IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ protein or fragments thereof.
  • the antibodies are specific for at least one biomarker from each neural cell type.
  • the composition of biomarkers is diagnostic of neural injury, damage and/or neural disorders.
  • the antibodies bind to: IL-I, IL-l ⁇ , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN- ⁇ , MCP-I, IP-10, MIP-Ib, MIP-3 ⁇ , TNF- ⁇ , VEGF, TIMP-I, ⁇ -NGF, CINC-2, Leptin, MCP-I, LIX and TGF- ⁇ protein or fragments thereof.
  • the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention.
  • the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody.
  • the antibody is attached to a solid support.
  • the antibody may be a monoclonal antibody.
  • the detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
  • test serum is reacted with a solid phase reagent having a surface -bound antigen obtained by the methods of the present invention.
  • the reagent After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
  • T he reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96- well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with bio tiny lated antigen(s).
  • the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a marker detected in a sample is a diagnostic amount consistent with a diagnosis of neural injury, degree of severity of the injury, subcellular localization, neuronal disorder and/or effect of treatment on the patient.
  • a kit comprises: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent is suitable for binding a marker, and (b) instructions to detect the marker or markers by contacting a sample with the adsorbent and detecting the marker or markers retained by the adsorbent.
  • the kit may comprise an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluant allows detection of the markers using gas phase ion spectrometry.
  • kits can be prepared from the materials described above, and the previous discussion of these materials (e.g., probe substrates, adsorbents, washing solutions, etc.) is fully applicable to this section and will not be repeated.
  • the kit may comprise a first substrate comprising an adsorbent thereon (e.g., a particle functionalized with an adsorbent) and a second substrate onto which the first substrate can be positioned to form a probe which is removably insertable into a gas phase ion spectrometer.
  • the kit may comprise a single substrate which is in the form of a removably insertable probe with adsorbents on the substrate.
  • the kit may further comprise a pre-fractionation spin column (e.g., Cibacron blue agarose column, anti-HSA agarose column, size exclusion column, Q-anion exchange spin column, single stranded DNA column, lectin column, etc.).
  • a pre-fractionation spin column e.g., Cibacron blue agarose column, anti-HSA agarose column, size exclusion column, Q-anion exchange spin column, single stranded DNA column, lectin column, etc.
  • the kit can further comprise instructions for suitable operational parameters in the form of a label or a separate insert.
  • the kit may have standard instructions informing a consumer how to wash the probe after a sample is contacted on the probe.
  • the kit may have instructions for pre-fractionating a sample to reduce complexity of proteins in the sample.
  • the kit may have instructions for automating the fractionation or other processes.
  • TBI patients Patients > 15 years of age with severe TBI defined as having a sum GCS of ⁇ 8 on the post-resuscitation admission neurological examination and requiring a ventriculostomy catheter for clinical management.
  • control patients Patients > 15 years of age requiring an intrathecal catheter or lumbar puncture to rule out meningitis or a subarachnoid bleed or required as part of their routine anesthetic or surgical management (e.g. endovascular aortic aneurysm stent repair, selected orthopedic procedure).
  • CSF samples from TBI patients CSF will be obtained from all consented subjects in whom a ventriculostomy catheter is placed as part of their standard medical care after TBI. The first CSF sample will be obtained at the time the ventriculostomy catheter is placed. Timed CSF samples (1 ml) will be collected at the time of the insertion of the ventriculostomy catheter (usually this will be within the first 6 hr after injury), and then at 6hr, 12hr, 18hr, 24hr and then every 24hr until the catheter is no longer needed for clinical management up to a maximum of 7 days. Pooled CSF, collected in the drainage bag as a result of CSF removal to control intracranial pressure (ICP) (per routine clinical care), will be collected on the same schedule as the timed CSF samples. The volume of CSF removed will be recorded.
  • ICP intracranial pressure
  • Blood samples will be collected from TBI or control groups and archived Blood samples will be collected on the same schedule as CSF samples. When ever possible, blood samples will be obtained at the same time blood for routine laboratory studies is obtained. If a patient has no laboratory studies ordered but has vascular access (venous or arterial line in place), the study sample will be drawn from the patient's vascular access catheter. Additionally, blood samples will be collected from the jugular bulb venous catheter (used clinically to monitor brain oxygenation). The total amount of blood volume sampled will approximately be 55 ml over the entire study.
  • Cytokine ELISAs Commercial sandwich ELISA' s for cytokines (e.g. IL-6, IL-10) are used. ELISAs will be performed according to manufacturer's instructions. For example, IL-6 assay kits are from Biosource (#KAC1261 for normal sensitivity, KHC0061 for high sensitivity). A 96-well microplate spectrophotometer (Gemini XS, Molecular Devices) will be used for recording all readings. Signal optimization will be conducted with the protein or glutamate and diluted with normal human CSF fluid, in each case. The standard curve will be run for each independent analysis of human CSF or serum samples. Standard curve fitting always employs a correlation coefficient of 0.95 or larger.
  • IL-6 assay kits are from Biosource (#KAC1261 for normal sensitivity, KHC0061 for high sensitivity).
  • a 96-well microplate spectrophotometer (Gemini XS, Molecular Devices) will be used for recording all readings. Signal optimization will be conducted with the protein
  • Example 2 Evaluation of rat IL-6 and IL-10 immunological parameters following exposure to methamphetamine and MDMA
  • Abuse Club drugs abuse of psychostimulant such as Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA ) abuse represents an immediate problem for the user and a potential long range problem as well.
  • METH Methamphetamine
  • MDMA 3,4-methylenedioxymethamphetamine
  • METH Methamphetamine
  • MDMA 3,4-methylenedioxymethamphetamine
  • TBI and naive samples serum collection A controlled cortical impact (CCI) device was used to model TBI in male Sprague-Dawley rats as described elsewhere. TBI injury was performed on 4 animals, each mounted in a stereotactic frame and impacted in the right cortex (ipsilateral) with a 5-mm diameter aluminum impactor tip at a velocity of 3.5 m/sec to a depth of 1.6-mm. Simultaneously, four na ⁇ ve control animals were kept under the same environmental conditions, but did not receive an impact injury. Serum collection was performed similar to the METH MDMA Saline groups described else where.
  • IL-6, IL-IO ELISA IL-6 and IL-IO levels were determined by enzyme immunoassay (Bender MedSystems). The procedure described by the manufacturer was followed. Sera from blood collected 20 rat animals divided into the five different groups
  • IL-6 and IL-10 showed significant increase in both TBI and drug abuse models
  • IL-6 is a pro-inflammatory cytokine secreted by T cells and macrophages to stimulate immune response to trauma, especially burns or other tissue damage leading to inflammation.
  • IL-10 is a pleiotropic cytokine that can exert either immunosuppressive or immunostimulatory effects on a variety of cell types.
  • the inhibitory effects of IL-10 on mouse THl cytokine synthesis was found to be indirect and due to the inhibitory effects of mouse IL-10 on the accessory function and antigen-presenting capacity of monocyte/macrophages .
  • Example 2 Elevation of pro-inflammatory and anti-inflammatory Cytokines in Rat Serum after Acute Methamphetamine Treatment and Traumatic Brain Injury
  • TBI Animal model A controlled cortical impact (CCI) device was used to model TBI in male Sprague-Dawley rats as described elsewhere (Pike et al., 1998, Neuroreport 9, 2437-42). TBI injury was performed on seven animals, each mounted in a stereotactic frame and impacted in the right cortex (ipsilateral) with a 5 -mm diameter aluminum impactor tip at a velocity of 3.5 m/sec to a depth of 1.6-mm. Seven na ⁇ ve control animals were kept under the same environmental conditions, but did not receive an impact injury. Na ⁇ ve and injured animals (48 hrs post injury) were sacrificed by decapitation.
  • CCI cortical impact
  • Densitometry Evaluation Densitometric quantification of the sample array films was performed using Epson expression 8836XL high-resolution flatbed scanner and NIH ImageJ densitometry software (version 1.6, NIH, Bethesda, MD). The densitometry values were evaluated for statistical significance with SigmaStat software (Version 2.03, Systat Software Inc.). All data presented are expressed as mean ⁇ SEM. Student's t-test was used to draw comparisons between intensities in the treated group (METH and TBI) vs. control (saline) group.
  • IL-6 and IL-10 ELISA Procedure IL-6 and IL-10 levels were determined by an enzyme linked immunoassay purchased from Bender MedSystems (Vienna, Austria) which was applied to validate the protein chip data. Serum cytokine levels were measured from 12 rats (TBI, Na ⁇ ve, METH, MDMA and control saline) according to the manufacturer instructions. The cut off value of serum IL-6 and IL-10 levels are 12 pg/ml and 1.47 pg/ml respectively. The values given are quoted in pg/ml. [0158] Results
  • IL-6 and IL-10 Levels Quantitation by ELISA In order to check the reliability of the relative differences of cytokines observed by using the Cytokine Antibody Array, we quantified IL-6 and IL-10 cytokines; two cytokines that showed upregulation in both TBI and acute METH insult by a conventional ELISA, as described above.
  • cytokine data revealed an elevation in serum IL- l ⁇ , IL-6 and IL-10 in both TBI and acute METH treatment.
  • IL-6 and IL-10 levels which were quantified by sandwich ELISA confirmed the cytokine antibody array data as shown in Figures 6A-6B.
  • Our TBI data showing elevated IL-6 and IL-10 are in concert with human TBI studies which show a marked elevation of both pro-inflammatory and anti-inflammatory cytokines including IL-I ⁇ , IL-6, IL-8, TNF- ⁇ and IL-10 which are detected at different time points in time course studies. Cytokine differential changes are detected in serum or CSF and are related to BBB disruption.
  • IL-6 it is a pleiotropic cytokine that regulates immune response mechanism and is produced not only by the immune cells but epithelial cells; IL- l ⁇ is a pro-inflammatory cytokine produced by macrophages and dendritic cells. Serum elevation of these two cytokines may be indicative of the pro -inflammatory environment at the injured area which is infiltrated by peripheral immune cells due to BBB leakage.
  • IL-IO was shown to be elevated after TBI; IL-IO is an anti-inflammatory cytokine that inhibits several macrophages functions including pro-inflammatory cytokine production.
  • IL-10 Although generally considered an immunosuppressive molecule; IL-10 exhibits some immunostimulatory properties as shown in different studies. In this context, several studies have shown that IL-10 enhances the function of natural killer cells which lead to antigen presentation by antigen presenting cells. Whether IL-10 is acting as an immuno- suppressive agent attenuating the inflammatory actions of IL- l ⁇ and IL-6 cytokines or as a stimulatory to the immune response, its serum elevation would be of similar pattern detected in the TBI insult so as to quell the damage of the potent secreted pro-inflammatory cytokines.
  • cytokine data are indicative of an immunomodulation process occurring post acute METH exposure and TBI brain insults.
  • the dual elevation of the proinflammatory cytokines IL- l ⁇ and IL-6 indicate a potentially harmful neuron inflammation response to neuronal cell death in METH abuse or TBI.
  • Future work would necessitate the evaluation of cytokines levels at different time points for a better understanding of immune system dynamics involvement in the area of drug abuse and TBI.
  • pro-inflammatory cytokines are present after acute METH abuse indicate a possible use of different anti-inflammatory agents; this indeed, presents a novel strategy in treating METH induced toxicity.

Abstract

Drug abuse (METH and MDMA) animals are showing a pro-inflammatory phenotype similar to that of TBI. Detection of cytokines in neurological injury is shown. Methods of treatment of neurological chemical injury include the use of an anti-inflammatory agents due to the elevation of inflammatory related cytokines.

Description

DIAGNOSIS AND TREATMENT OF NEUROLOGICAL INFLAMMATION
FIELD OF THE INVENTION
[0001] The invention provides for the detection and diagnosis of chemical neurological injury. Detection of cytokines allows for methods of treating and monitoring the course of treatment in patients with neural injury.
BACKGROUND
[0002] Methamphetamine (Meth) also known as ice, crank, speed, crystal meth is an addictive psycho stimulant belonging to the amphetamine family, which was originally introduced as an appetite suppressant and a treatment for attention deficit disorder. It is the most widely spread illicit drug because it is easy to prepare and cheap to obtain. Methamphetamine can be smoked, inhaled, or injected leading to a variety of social behaviors with a feeling of euphoria and increased self esteem, with the effects lasting up to 15 hours. Amphetamines are sympathomemetics that act by increasing the amount of dopamine, norepinephrine, and epinephrine neurotransmitters available in the brain. As a result, the user may experience a heightened sense of awareness and paranoia. The average methamphetamine dose may cause the user to experience mydriasis (dilated pupils), hypertension, tachycardia (increased heart rate), and hyperthermia. Other indicators of methamphetamine use include auditory hallucination, visual hallucinations often involving bugs, agitation, insomnia, anxiety, nausea, and vomiting. Upon methamphetamine overdose, psychosis, myocardial infarction, seizures, and even death may occur. [0003] Methamphetamine is a psychostimulant that is known to mediate addictive behavior by acting on the monoaminergic system leading to an increase in the dopamine (DA) and serotonin (5-UT) levels in certain brain regions. Several human and non-human primate studies have indicated that the methamphetamine monoaminergic effect can lead to neurotoxic effects, which are associated with neurocognitive impairments involving memory deficits (nonspatial and working memory). Long-term methamphetamine use induces dopaminergic and serotonergic axonal terminal damage that is coupled with neuronal degeneration of specific population of neocortical neurons, and is attributed to the lipophilic nature of methamphetamine, which facilitates crossing of the blood brain barrier (BBB) and access to different brain regions. Upon entering the monoaminergic system, meth binds to the plasmalemal dopamine transporter (DAT) and alters its function by blocking the re -uptake of DA and overloading the synapse. Methamphetamine also diffuses via the DAT into the neuronal terminal where it acts as a substrate for a number of neuronal structures, including the dopamine vesicular transporter (VMAT) that contributes to increased cytosolic DA. Afterward, neuronal function is altered leading to a reduction in DAT activity and the dysfunction of tyrosine hydroxylase (TH) and dopamine vesicular transporter. Furthermore, the oxidizing environment of the cytosol can lead to DA oxidation leading to the generation of nitrogen, oxygen, and metabolic reactive species that trigger dopaminergic terminal degeneration and subsequently cause necrotic cell death. In addition, it has been shown that methamphetamine can diffuse into other neuronal organelles, such as the mitochondria where it causes perturbation in the mitochondrial electron gradient leading to mitochondrial mediated apoptotic cell death. Oxidative stress mediated injury is exacerbated by increased glutamate levels, which in turn can activate NMDA receptors producing more reactive oxygen species leading toward excitotoxicity. Neuronal degeneration is not confined to the monoaminergic system but can include other neocortical cells in a dopamine independent pathway. An immortalized neuronal cell line when treated with methamphetamine exhibited an apoptotic cell death phenotype.
[0004] A need therefore, exists in the art for diagnosis of neurological monitoring of harmful brain damage due to inflammation and to treat such inflammation with available anti-inflammatory drugs or procedures.
SUMMARY
[0005] A method of diagnosing neurological injury in a patient comprises identifying cytokines and cytokine levels in a sample from an injured patient, wherein the cytokines detected are elevated or decreased as compared to normal individuals which can reflect on the altered immune status of the inflicted patient.
[0006] In a preferred embodiment, the cytokines detected comprise: IL-I, IL- lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β. The cytokine levels can be correlated to known biomarkers of neurological injury. Examples include, but not limited to: neural proteins, such as for example, axonal proteins - NF-200 (NF-H), NF-160 (NF-M), NF-68 (NF-L); amyloid precursor protein; dendritic proteins - alpha-tubulin (P02551), beta-tubulin (PO 4691), MAP- 2A/B, MAP-2C, Tau, Dynamin-1 (P21575), Dynactin (Q13561), P24; somal proteins - UCH- Ll (Q00981), PEBP (P31044), NSE (P07323), Thy 1.1, Prion, Huntington; presynaptic proteins - synapsin-1, synapsin-2, alpha-synuclein (p37377), beta-synuclein (Q63754), GAP43, synaptophysin, synaptotagmin (P21707), syntaxin; post-synaptic proteins - PSD95, PSD93, NMDA-receptor (including all subtypes); demyelination biomarkers - myelin basic protein (MBP), myelin proteolipid protein; glial proteins - GFAP (P47819), protein disulfide isomerase (PDI - P04785); neurotransmitter biomarkers - cholinergic biomarkers: acetylcholine esterase, choline acetyltransferase; dopaminergic biomarkers - tyrosine hydroxylase (TH), phospho-TH, DARPP32; noradrenergic biomarkers - dopamine beta- hydroxylase (DbH); serotonergic biomarkers - tryptophan hydroxylase (TrH); glutamatergic biomarkers - glutaminase, glutamine synthetase; GABAergic biomarkers - GABA transaminase (4-aminobutyrate-2-ketoglutarate transaminase [GABAT]), glutamic acid decarboxylase (GAD25, 44, 65, 67); neurotransmitter receptors - beta-adrenoreceptor subtypes, (e.g. beta (2)), alpha-adrenoreceptor subtypes, (e.g. (alpha (2c)), GABA receptors (e.g. GABA(B)), metabotropic glutamate receptor (e.g. mGluR3), NMDA receptor subunits (e.g. NR1A2B), Glutamate receptor subunits (e.g. GluR4), 5-HT serotonin receptors (e.g. 5- HT(3)), dopamine receptors (e.g. D4), muscarinic Ach receptors (e.g. Ml), nicotinic acetylcholine receptor (e.g. alpha-7); neurotransmitter transporters - norepinephrine transporter (NET), dopamine transporter (DAT), serotonin transporter (SERT), vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), glutamate transporter (e.g. GLTl), vesicular acetylcholine transporter, choline transporter (e.g. CHTl); other protein biomarkers include, but not limited to vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2, MMP9. [0007] Other examples of biomarkers of neurological injury to which cytokine biomarkers or levels of cytokines can be correlated with the detection of one or more of: Axonal Proteins: α II spectrin ( and SPDB)-I, NF-68 (NF-L) - 2, Tau - 3, α II, III spectrin, NF- 200 (NF-H), NF- 160 (NF-M), Amyloid precursor protein, α internexin; Dendritic Proteins: beta Ill-tubulin - 1, p24 microtubule-associated protein - 2, alpha-Tubulin (P02551), beta-Tubulin (P04691), MAP-2A/B - 3, MAP-2C -3, Stathmin - 4, Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000), Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-Ll (Q00981) - 1, Glycogen phosphorylase-BB - 2, PEBP (P31044), NSE (P07323), CK-BB (P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g. 14-3-3- epsolon (P42655)), SM22-α, Calgranulin AB, alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural nuclear proteins: NeuN - 1, S/G(2) nuclear autoantigen (SG2NA), Huntingtin; Presynaptic Proteins: Synaptophysin - 1, Synaptotagmin (P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsinl (Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43, Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMPl, 2, Amphiphysin -1 (NP_001626), Amphiphysin -2 (NP_647477); Post-Synaptic Proteins: PSD95 - 1, NMDA-receptor (and all subtypes) -2, PSD93, AMPA-kainate receptor (all subtypes), mGluR (all subtypes), Calmodulin dependent protein kinase II (CAMPK)- alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP; Myelin- Oligodendrocyte: Myelin basic protein (MBP) and fragments, Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin associated protein (MAG), Oligodendrocyte NS-I protein; Glial Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase (PDI) - P04785, Neurocalcin delta, SlOObeta; Microglia protein Biomarkers: Ibal, OX-42, OX-8, OX-6, ED-I, PTPase (CD45), CD40, CD68, CDl Ib, FractaMne (CX3CL1) and Fractalkine receptor (CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin, SCGlO; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K, Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35; Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus: CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin receptors ( OX-IR and 0X-2R)- appetite, Orexins (hypothalamus - specific peptides); Corpus callosum: MBP, MOG, PLP, MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin, Rhes (Ras homolog enriched in striatum); Peripheral ganglia: Gadd45a; Peripherial nerve fiber(sensory + motor): Peripherin, Peripheral myelin protein 22 (AAH91499); Other Neuron-specific proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin (NC), a neuron- specific EF-hand Ca2+ -binding protein, Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin; Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B), Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GIuRl, GluR4), beta-adrenoceptor subtypes (e.g. beta(2)), Alpha- adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g. GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4), Muscarinic Ach receptors (e.g. Ml), Nicotinic Acetylcholine Receptor (e.g. alpha-7); Neurotransmitter Transporters: Norepinephrine Transporter (NET), Dopamine transporter (DAT), Serotonin transporter (SERT), Vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLTl), Vesicular acetylcholine transporter, Vesicular Glutamate Transporter 1, [VGLUTl; BNPI] and VGLUT2, Choline transporter, (e.g. CHTl); Cholinergic Biomarkers: Acetylcholine Esterase, Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers: Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers: Phenylethanolamine N-methyltransferase (PNMT); Serotonergic Biomarkers: Tryptophan
Hydroxylase (TrH); Glutamatergic Biomarkers: Glutaminase, Glutamine synthetase;
GABAergic Biomarkers: GABA transaminase [GABAT]), GABA-B-R2.
[0008] In another preferred embodiment, a patient can be treated with anti-inflammatory agents (e.g. steroidal or non-steroidal) and/or anti-cytokine antibodies to decrease the inflammation. Anti-inflammatory agents are well know to one of ordinary skill in the art.
[0009] In another preferred embodiment, a composition of markers for brain injury comprise: IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70),
IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-
1, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β.
[0010] In another preferred embodiment, antibodies specific for cytokines comprise IL-I,
IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13,
Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-
NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β.
[0011] In another preferred embodiment, the sample is selected from the group consisting of saliva, sputum, blood, blood plasma, serum, urine, tissue, cells, and liver.
[0012] In another preferred embodiment, a method of treating a patient suffering from neurological disorders comprising measuring the cytokine levels in the patients and treating the patient with anti-inflammatory agents.
[0013] In a preferred embodiment, a patient is treated with agents that decrease cytokine levels identified as diagnostic of neurological damage as compared to a normal individual.
Preferably, decreasing the cytokine levels decreases neurological injury.
[0014] In another preferred embodiment, a method of monitoring effectiveness of treatment of neural injury comprises measuring cytokine levels and/or total protein in a biological sample obtained from said subject, wherein detected levels of cytokines and/or total protein compared to normal subjects is indicative of the effectiveness of treatment of neurological injury.
[0015] In another preferred embodiment, decreased levels of cytokines and/or total protein in a biological sample obtained from said subject, as compared to levels detected prior to treatment in the same patient, is prognostic of the effectiveness of treatment.
[0016] In another preferred embodiment, a method of identifying the severity of neurological injury course in a patient, comprising measuring cytokines and/or total protein in a biological sample obtained from said subject, wherein an elevated level of cytokines and/or total protein compared to normal subjects is indicative of the severity of neurological injury.
[0017] In a preferred embodiment, decreased levels of cytokines and/or total protein in a biological sample obtained from said subject, as compared to levels detected prior to treatment in the same patient, is prognostic of the effectiveness of treatment.
[0018] In another preferred embodiment, the sample is selected from the group consisting of cerebrospinal fluid, blood, blood plasma, serum, urine, tissue, cells, and organs.
[0019] In another preferred embodiment, cytokine levels and/or total protein are detected using an immunoassay. For example, ELISA, RIA and the like.
[0020] In another preferred embodiment, the cytokine levels and/or total protein are detected using a biochip array. In one aspect, the biochip array is a protein chip array. In another aspect the biochip array is a nucleic acid array. The biochip can comprise one or more antibodies specific for cytokines; single or double stranded nucleic acids; proteins, peptides or fragments thereof; amino acid probes; phage display libraries; one or more cytokines and/or total proteins.
[0021] In another preferred embodiment, a kit comprises IL-I, IL-lβ, IL-2, IL-4, IL-5,
IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ,
MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I,
LIX and TGF- β, peptides, fragments or antibodies thereof.
[0022] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] There is shown in the drawings embodiments, which are presently preferred, it being understood, however, that the invention can be embodied in other forms without departing from the spirit or essential attributes thereof.
[0024] Figures IA and IB are graphs showing the results from immunological (ELISA) assays for cytokines IL-6 (Figure IA) and IL-10 (Figure IB) in serum showing significant increases after Meth and MDMA acute overdose and traumatic brain injury. Compared to saline control animals, both Meth and MDMA showed statistically significant increases in IL-6 at 24 hours after treatment (p values of 0.0043 and 0.0258 respectively). Meth also showed a statistically significant increase in IL-10 at 24 hours after treatment (p value of 0.0003). We compared those results to rat brain trauma (1.6 mm severe control cortical impact), which showed a similar cytokine increase to that of Meth at 48 hours after injury. [0025] Figures 2A and 2B are scans of Western blots showing spectrin structural protein degradation indicative of induced neurotoxicity in TBI and acute model of methamphetamine (40mg/kg). αll-spectrin proteolysis in rat brain 24 hours following meth administration. For saline and meth treatments, n=6. For naϊve and TBI (1.6 mm), n=3. The αll-spectrin parent protein as well as spectrin breakdown products at 150, 145 and 120 kDa were observed. B- actin was shown as an internal standard for loading equivalence. Figure 2A are the results from cortex. Figure 2B are the results from hippocampus. These data are indicative of CNS injury and neuronal cell death reflecting a marked similarity between acute Meth injury and mechanical brain injury.
[0026] Figure 3 is a graph showing cytokine IL-6 ELISA assay of clinical TBI samples. CSF and serum samples were collected from TBI patients at the indicated time points following injury and were compared with non-trauma CSF and serum control samples (n values as indicated).
[0027] Figure 4 is a graph showing changes in rat weight after acute METH administration and TBI insult. For saline treatments (white bars), n=4, acute METH treatments (Dashed bars), n=4 and TBI rats (Grey bars), n= 4. Rat weight decreases significantly following the 24 hrs drug administration which reached approximately 12% of the total body weight. This weight loss was not significant in TBI rats. Results are expressed as mean ± SEM gms and * marks above bars represent the statistical significance between the groups at the level of P<0.05. Student's t tests was applied for statistical significance (p<0.005 for each).
[0028] Figures 5A-5B show the serum analysis of the rat cytokine antibody array. Serum analysis of the rat cytokine antibody array revealed a correlation between increasing levels of certain cytokines with the two brain insults (acute METH administration and TBI). A panel of 19 secreted cytokines was checked for any changes in the serum of control saline rats, acute METH administration and TBI insults by using the RayBiotec cytokine array kit. Figure 5A. A representative cytokine blot is shown from the saline control, METH treated and TBI samples. The boxes bordering differential spots on the blots demonstrate the cytokines that are up-regulated which included IL- lβ, IL-6 and IL-10 compared with the saline control serum. Figure 5B. The actual cytokine array map from RayBio™ is also provided; it detects GM-CSF, IFN-γ, IL-l-β, IL-4, TIMP-I, β-NGF, CINC-2, Leptin, MIP- 3α, MCP-I, LIX, IL-10, TNF-α, VEGF and IL-6.
[0029] Figures 6A-6B are graphs showing altered cytokines analysis in the rat serum after acute meth administration and TBI Insult. Graphical representation of the up-regulated cytokines following the acute METH treatment (grey bars) compared to saline control (white bars), significant changes were observed (IL- lβ, IL-6 and IL-IO cytokines) shown in Figure 6A. In Figure 6B, graphical representation of the up-regulated cytokines following the TBI insult (dashed bars) compared to saline control, significant changes were observed (IL- lβ, IL- 6 and IL-10 cytokines). Results are expressed as mean +/- SEM arbitrary densitometric units and * marks above bars represent the statistical significance between the groups at the level of P<0.05. Student's t-tests was applied for statistical significance (p<0.005 for each). [0030] Figures 7A-7B are graphs showing ELISA quantitation of serum IL-6 and IL-10 cytokines after acute METH administration and TBI insult. Serum concentrations of respective IL-6 and IL-10 after saline treatment (white bars) acute METH treatment (grey bars) and TBI rats (dashed bars). IL-6 and IL-10 levels were increased significantly in similar manners after acute METH treatments and TBI insult. Results are expressed as mean +/- SEM pg/ml and * marks above bars represent the statistical significance between the groups at the level of P<0.05. Student's t-tests was applied for statistical significance (p<0.005 for each).
DETAILED DESCRIPTION
[0031] The present invention provides methods of diagnostics and treatment of acute chemical brain injury including drug abuse brain injury due to acute exposure to methamphetamine (e.g., overdose). The methods are utilized for any form of neurological injury (e.g., chemical injury or mechanical).
Definitions
[0032] As used herein, the term "injury or neural injury" is intended to include a damage which directly or indirectly affects the normal functioning of the CNS and PNS. For example, the injury can be damage to retinal ganglion cells; a traumatic brain injury; a stroke related injury; a cerebral aneurism related injury; a spinal cord injury, including monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia; a neuroproliferative disorder or neuropathic pain syndrome. Neurological or neural injury includes damage to the brain due to drugs. Other neurological injuries include: acute spinal cord trauma, spinal cord compression, spinal cord hematoma, cord contusion (these cases are usually traumatic, such as motorcycle accidents or sports injuries); nerve compression, the most common condition being a herniated disc causing sciatic nerve compression, neuropathy, and pain; but also including cervical disc herniation, causing nerve compression in the neck; carpal tunnel syndrome (non-RA); acute or chronic spinal cord compression from cancer (this is usually due to metastases to the spine, such as from prostate, breast or lung cancer); autoimmune disease of the nervous system; and demyelinating diseases, the most common condition being multiple sclerosis. Another form of brain injury include chemical brain injury (CBI) which may be induced due to the exposure of different club drugs including methamphetamine, cocaine, MDMA and these drugs are accessible to the brain and can induce some form of brain injury. Data collected in the area of drug abuse and particularly acute methamphetamine we have shown that, this club drug induces cytoskeletal damage of brain protein which can lead to irreversible brain damage.
[0033] Examples of CNS injuries or disease include TBI, stroke, concussion (including post-concussion syndrome), cerebral ischemia, neurodegenerative diseases of the brain such as Parkinson's disease, Dementia Pugilistica, Huntington's disease and Alzheimer's disease, Creutzfeldt-Jakob disease, brain injuries secondary to seizures which are induced by radiation, exposure to ionizing or iron plasma, nerve agents, cyanide, toxic concentrations of oxygen, neurotoxicity due to CNS malaria or treatment with anti-malaria agents, trypanosomes, malarial pathogens, and other CNS traumas.
[0034] As used herein, the term "Traumatic Brain Injury" is art recognized and is intended to include the condition in which, a traumatic blow to the head causes damage to the brain, often without penetrating the skull. Usually, the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF).
[0035] As used herein, "cytokine" is used generally. The term includes: lymphokine (cytokines produced by lymphocytes), monokine (cytokines produced by monocytes), chemokine (cytokines with chemotactic activities), and interleukin (cytokines produced by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action), or in some instances on distant cells (endocrine action).
[0036] "Pro-inflammatory cytokines" are cytokines produced predominantly by activated immune cells such as microglia and are involved in the amplification of inflammatory reactions. These include IL-I, IL-6, TNF-α, and TGF-β.
[0037] "Anti-inflammatory cytokines" belong to the T cell-derived cytokines and are involved in the down -regulation of inflammatory reactions. The anti-inflammatory cytokines are a series of immunoregulatory molecules that control the proinflammatory cytokine response. Cytokines act in concert with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response. Major anti-inflammatory cytokines include interleukin (IL)-I receptor antagonist, IL-4, IL-6, IL-IO, IL-Il, and IL-13. Specific cytokine receptors for IL-I, tumor necrosis factor-alpha, and IL- 18 also function as proinflammatory cytokine inhibitors.
[0038] The phrase "differentially present" refers to differences in the quantity and/or the frequency of a marker present in a sample taken from patients having for example, neural injury as compared to a control subject. For example, a marker can be a polypeptide which is present at an elevated level or at a decreased level in samples of patients with neural injury compared to samples of control subjects. Alternatively, a marker can be a polypeptide which is detected at a higher frequency or at a lower frequency in samples of patients compared to samples of control subjects. A marker can be differentially present in terms of quantity, frequency or both.
[0039] A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is statistically significantly different from the amount of the polypeptide in the other sample. For example, a polypeptide is differentially present between the two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other. [0040] Alternatively or additionally, a polypeptide is differentially present between the two sets of samples if the frequency of detecting the polypeptide in samples of patients' suffering from neural injury and/or neuronal disorders, is statistically significantly higher or lower than in the control samples. For example, a polypeptide is differentially present between the two sets of samples if it is detected at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% more frequently or less frequently observed in one set of samples than the other set of samples. [0041] "Diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
[0042] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).
[0043] A "diagnostic amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of neural injury and/or neuronal disorder. A diagnostic amount can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).
[0044] A "control amount" of a marker can be any amount or a range of amount which is to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a person without neural injury and/or neuronal disorder. A control amount can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).
[0045] "Sample" is used herein in its broadest sense. A sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
[0046] "Neural (neuronal) defects, disorders or diseases" as used herein refers to any neurological disorder, including but not limited to neurodegenerative disorders (Parkinson's; Alzheimer's) or autoimmune disorders (multiple sclerosis) of the central nervous system; memory loss; long term and short term memory disorders; learning disorders; autism, depression, benign forgetfulness, childhood learning disorders, close head injury, and attention deficit disorder; autoimmune disorders of the brain, neuronal reaction to viral infection; brain damage; depression; psychiatric disorders such as bi-polarism, schizophrenia and the like; narcolepsy/sleep disorders(including circadian rhythm disorders, insomnia and narcolepsy); severance of nerves or nerve damage; severance of the cerebrospinal nerve cord (CNS) and any damage to brain or nerve cells; neurological deficits associated with AIDS; tics (e.g. Giles de Ia Tourette's syndrome); Huntington's chorea, schizophrenia, traumatic brain injury, tinnitus, neuralgia, especially trigeminal neuralgia, neuropathic pain, inappropriate neuronal activity resulting in neurodysthesias in diseases such as diabetes, MS and motor neurone disease, ataxias, muscular rigidity (spasticity) and temporomandibular joint dysfunction; Reward Deficiency Syndrome (RDS) behaviors in a subject.
Biomarkers
[0047] In a preferred embodiment, a composition of biomarkers comprises IL-I, IL- lβ,
IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM-
CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2,
Leptin, MCP-I, LIX and TGF-β.
[0048] In another preferred embodiment, antibodies specific for biomarkers of neurological injury comprise antibodies specific for IL-I, IL- lβ, IL-2, IL-4, IL-5, IL-6, IL-8,
IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10,
MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β.
[0049] In a preferred embodiment, a method of diagnosing neurological injury in a patient comprises identifying cytokines and cytokine levels in a sample from an injured patient, wherein the cytokines detected are elevated as compared to normal individuals.
Examples of detected cytokines include, but not limited to: IL-I, IL- lβ, IL-2, IL-4, IL-5, IL-
6, IL-8, IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I,
IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and
TGF-β.
[0050] In another preferred embodiment, the sample is selected from the group consisting of saliva, sputum, blood, blood plasma, serum, urine, tissue, cells, and liver.
[0051] In another preferred embodiment, the cytokine levels are compared to known biomarkers of neurological injury. For example, α II spectrin, spectrin break down products,
MBP, etc.
[0052] In another preferred embodiment, a method of treating a patient suffering from neurological disorders comprises measuring the cytokine levels in the patients and treating the patient with compounds that decrease the cytokine levels to levels indicative of a normal individual. Preferably, the patient is treated with anti-inflammatory agents and/or agents that decrease inflammatory cytokine production.
[0053] In a preferred embodiment, treatment of a patient comprises administering one or more anti-inflammatory agent, anti-inflammatory cytokines, cytokine inhibitors such as for example, receptor blockers.
[0054] In another preferred embodiment, a method of monitoring effectiveness of treatment of neural injury comprises measuring cytokine levels and/or total protein in a biological sample obtained from said subject, wherein detected levels of cytokines and/or total protein compared to normal subjects is indicative of the effectiveness of treatment of neurological injury.
[0055] In another preferred embodiment, decreased levels of cytokines and/or total protein in a biological sample obtained from said subject, as compared to levels detected prior to treatment in the same patient, is prognostic of the effectiveness of treatment. [0056] In another preferred embodiment, a method of identifying the severity of neurological injury course in a patient, comprises measuring cytokines and/or total protein in a biological sample obtained from said subject, wherein an elevated level of cytokines and/or total protein compared to normal subjects is indicative of the severity of neurological injury. [0057] In another preferred embodiment, treatment of a patient suffering from neurological injury comprises administration of one or more antibodies to IL-I, IL- lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β protein or fragments thereof. The treatment can be in conjunction with administration of one or more ant-inflammatory agents which include both steroidal and non-steroidal agents, analgesics and the like.
[0058] In one preferred embodiment, the cytokine biomarkers of neurological injury are correlated to other biomarkers of neurological injury. Examples include, but not limited to: neural proteins, such as for example, axonal proteins - NF-200 (NF-H), NF- 160 (NF-M), NF- 68 (NF-L); amyloid precursor protein; dendritic proteins - alpha-tubulin (P02551), beta- tubulin (PO 4691), MAP-2A/B, MAP-2C, Tau, Dynamin-1 (P21575), Dynactin (Q13561), P24; somal proteins - UCH-Ll (Q00981), PEBP (P31044), NSE (P07323), Thy 1.1, Prion, Huntington; presynaptic proteins - synapsin-1, synapsin-2, alpha- synuclein (p37377), beta- synuclein (Q63754), GAP43, synaptophysin, synaptotagmin (P21707), syntaxin; postsynaptic proteins - PSD95, PSD93, NMDA-receptor (including all subtypes); demyelination biomarkers - myelin basic protein (MBP), myelin proteolipid protein; glial proteins - GFAP (P47819), protein disulfide isomerase (PDI - P04785); neurotransmitter biomarkers - cholinergic biomarkers: acetylcholine esterase, choline acetyltransferase; dopaminergic biomarkers - tyrosine hydroxylase (TH), phospho-TH, DARPP32; noradrenergic biomarkers - dopamine beta-hydroxylase (DbH); serotonergic biomarkers - tryptophan hydroxylase (TrH); glutamatergic biomarkers - glutaminase, glutamine synthetase; GABAergic biomarkers - GABA transaminase (4-aminobutyrate-2-ketoglutarate transaminase [GABAT]), glutamic acid decarboxylase (GAD25, 44, 65, 67); neurotransmitter receptors - beta-adrenoreceptor subtypes, (e.g. beta (2)), alpha-adrenoreceptor subtypes,(e.g. (alpha (2c)), GABA receptors (e.g. GABA(B)), metabotropic glutamate receptor (e.g. mGluR3), NMDA receptor subunits (e.g. NR1A2B), Glutamate receptor subunits (e.g. GluR4), 5-HT serotonin receptors (e.g. 5-HT(3)), dopamine receptors (e.g. D4), muscarinic Ach receptors (e.g. Ml), nicotinic acetylcholine receptor (e.g. alpha-7); neurotransmitter transporters - norepinephrine transporter (NET), dopamine transporter (DAT), serotonin transporter (SERT), vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), glutamate transporter (e.g. GLTl), vesicular acetylcholine transporter, choline transporter (e.g. CHTl); other protein biomarkers include, but not limited to vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2, MMP9.
[0059] Other examples of biomarkers of neurological injury to which cytokine biomarkers or levels of cytokines can be correlated with the detection of one or more of: Axonal Proteins: α II spectrin ( and SPDB)-I, NF-68 (NF-L) - 2, Tau - 3, α II, III spectrin, NF- 200 (NF-H), NF- 160 (NF-M), Amyloid precursor protein, α internexin; Dendritic Proteins: beta Ill-tubulin - 1, p24 microtubule-associated protein - 2, alpha-Tubulin (P02551), beta-Tubulin (P04691), MAP-2A/B - 3, MAP-2C -3, Stathmin - 4, Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000), Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-Ll (Q00981) - 1, Glycogen phosphorylase-BB - 2, PEBP (P31044), NSE (P07323), CK-BB (P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g. 14-3-3- epsolon (P42655)), SM22-α, Calgranulin AB, alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural nuclear proteins: NeuN - 1, S/G(2) nuclear autoantigen (SG2NA), Huntingtin; Presynaptic Proteins: Synaptophysin - 1, Synaptotagmin (P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsinl (Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43, Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMPl, 2, Amphiphysin -1 (NP_001626), Amphiphysin -2 (NP_647477); Post-Synaptic Proteins: PSD95 - 1, NMDA-receptor (and all subtypes) -2, PSD93, AMPA-kainate receptor (all subtypes), mGluR (all subtypes), Calmodulin dependent protein kinase II (CAMPK)- alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP; Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments, Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin associated protein (MAG), Oligodendrocyte NS-I protein; Glial Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase (PDI) - P04785, Neurocalcin delta, SlOObeta; Microglia protein Biomarkers: Ibal, OX-42, OX-8, OX-6, ED-I, PTPase (CD45), CD40, CD68, CDl Ib, Fractalkine (CX3CL1) and Fractalkine receptor (CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin, SCGlO; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K, Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35; Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus: CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin receptors ( OX-IR and 0X-2R)- appetite, Orexins (hypothalamus - specific peptides); Corpus callosum: MBP, MOG, PLP, MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin, Rhes (Ras homolog enriched in striatum); Peripheral ganglia: Gadd45a; Peripherial nerve fiber(sensory + motor): Peripherin, Peripheral myelin protein 22 (AAH91499); Other Neuron- specific proteins: PH8 (S Serotonergic Dopaminergic, PEP- 19, Neurocalcin (NC), a neuron-specific EF-hand Ca2+ -binding protein, Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin; Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B), Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GIuRl, GluR4), beta-adrenoceptor subtypes (e.g. beta(2)), Alpha- adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g. GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4), Muscarinic Ach receptors (e.g. Ml), Nicotinic Acetylcholine Receptor (e.g. alpha-7); Neurotransmitter Transporters: Norepinephrine Transporter (NET), Dopamine transporter (DAT), Serotonin transporter (SERT), Vesicular transporter proteins (VMATl and VMAT2), GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLTl), Vesicular acetylcholine transporter, Vesicular Glutamate Transporter 1, [VGLUTl; BNPI] and VGLUT2, Choline transporter, (e.g. CHTl); Cholinergic Biomarkers: Acetylcholine Esterase, Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers: Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers: Phenylethanolamine N-methyltransferase (PNMT); Serotonergic Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers: Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA transaminase [GABAT]), GABA-B-R2. [0060] In another preferred embodiment, the amount of cytokine marker detected, for example, in μg/ml is diagnostic of the extent of damage or injury. Quantitation of each biomarker is described in the specification and in the Examples to follow. Assays include immunoassays (such as ELISA' s), spectrophotometry, HPLC, SELDI, biochips and the like. The quantitation of each as compared to a normal individual is diagnostic of the extent of injury. [0061] In another preferred embodiment, neurological damage in a subject is analyzed by (a) providing a biological sample isolated from a subject suspected of having a neurological injury; (b) detecting in the sample the presence or amount of at least one marker selected from one or more cytokines; and (c) correlating the presence or amount of the cytokines with the presence or type of neurological injury in the subject. After injury (neurological damage) to the nervous system (such as brain injury), the neural cell membrane is compromised, leading to the efflux of these neural proteins first into the extracellular fluid or space and to the cerebrospinal fluid and eventually in the circulating blood (as assisted by the compromised blood brain barrier) and other biofluids (e.g. urine, sweat, s saliva, etc.). Thus, suitable biological samples include, but not limited to such cells or fluid secreted from these cells. Obtaining biological fluids such as cerebrospinal fluid, blood, plasma, serum, saliva and urine, from a subject is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample. Thus, samples, which are biological fluids, are preferred for use in the invention. CSF, in particular, is preferred for detecting nerve damage in a subject as it is in immediate contact with the nervous system and is readily obtainable. [0062] A biological sample can be obtained from a subject by conventional techniques. For example, CSF can be obtained by lumbar puncture. Blood can be obtained by venipuncture, while plasma and serum can be obtained by fractionating whole blood according to known methods. Surgical techniques for obtaining solid tissue samples are well known in the art. For example, methods for obtaining a nervous system tissue sample are described in standard neuro-surgery texts such as Atlas of Neurosurgery: Basic Approaches to Cranial and Vascular Procedures, by F. Meyer, Churchill Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain Tumors, 1st ed., by David G.T. Thomas, WB Saunders Co., 1993; and Cranial Microsurgery: Approaches and Techniques, by L. N. Sekhar and E. De Oliveira, 1st ed., Thieme Medical Publishing, 1999. Methods for obtaining and analyzing brain tissue are also described in Belay et al., Arch. Neurol. 58: 1673-1678 (2001); and Seijo et al., J. Clin. Microbiol. 38: 3892-3895 (2000).
[0063] Any animal that expresses neural proteins, such as for example, those described above, can be used as a subject from which a biological sample is obtained. Preferably, the subject is a mammal, such as for example, a human, dog, cat, horse, cow, pig, sheep, goat, primate, rat, mouse and other vertebrates such as fish, birds and reptiles. More preferably, the subject is a human. Particularly preferred are subjects suspected of having or at risk for developing traumatic or non-traumatic nervous system injuries, such as victims of brain injury caused by traumatic insults (e.g. gunshots wounds, automobile accidents, sports accidents, shaken baby syndrome), ischemic events (e.g. stroke, cerebral hemorrhage, cardiac arrest), spinal cord injury, neurodegenerative disorders (such as Alzheimer's, Huntington's, and Parkinson's diseases; Prion-related disease; other forms of dementia, and spinal cord degeneration), epilepsy, substance abuse (e.g., from amphetamines, methamphetamine/Speed , Ecstasy/MDMA, or ethanol and cocaine), and peripheral nervous system pathologies such as diabetic neuropathy, chemotherapy-induced neuropathy and neuropathic pain, peripheral nerve damage or atrophy (ALS), multiple sclerosis (MS).
[0064] As described above, the invention provides the step of correlating the presence or amount of one or more cytokines and/or total protein with the severity and/or type of neural injury. The amount of a cytokines, total protein, neural proteins, peptides, fragments, derivatives or the modified forms, thereof, directly relates to severity of nerve tissue injury as more severe injury damages a greater number of nerve cells which in turn causes a larger amount of neural protein(s) to accumulate in the biological sample (e.g., serum, plasma, blood, CSF).
[0065] Cytokines and Inflammation: The concept that the brain is an immunologically privileged organ has been challenged by recent evidence that inflammatory-like processes and immune reactions occur in the CNS after a large variety of peripheral or local stimuli. Cytokines are expressed at very low levels in healthy brain tissue under physiologic conditions; however, they can be rapidly induced there after a variety of injuries. In particular, production of proinflammatory cytokines is an early cellular event subsequent to ischemic, traumatic, and excito toxic insults.
[0066] Proinflammatory cytokines, such as interleukin (IL)-lβ, IL-6, IL-10 and tumor necrosis factor (TNF)-α, produce detrimental effects on brain function. The effects are dependent on a number of factors, including local concentration of the cytokine at the site of synthesis, the type of target cells, the length of time the tissue is exposed to cytokines, and which receptor subtypes are involved. Microglia and astrocytes are the first cells to produce cytokines during epileptic activity, and they represent the main source of local cytokine production in brain. An additional source of production consists of blood monocytes, pervading the brain several hours after acute epileptic events.
[0067] Cytokines make up the fourth major class of soluble intercellular signaling molecules, alongside neurotransmitters, endocrine hormones, and autacoids. They possess typical hormonal activities: they are secreted by a single cell type, react specifically with other cell types (target cells) and regulate specific vital functions that are controlled by feedback mechanisms; they generally act at short range in a paracrine or autocrine (rather than endocrine) manner; they interact first with high-affinity cell surface receptors (distinct for each type or even subtype) and then regulate the transcription of a number of cellular genes by little understood second signals. This altered transcription (which can be an enhancement or inhibition) result in changes in cell behavior.
[0068] Target cells, on which cytokines transform their information signal, may be localized in any body compartment (sometimes a long distance from the site of secretion). Other type of these molecules act mostly on neighboring cells in the microenvironment where they have been released. These are characterized as local hormones and their secretion is brought about by autocrine (only the cell or organ of secretion is affected) or paracrine mechanisms. During the paracrine secretion some cytokines may escape cell binding and may spill over into general circulation via lymph or plasma. This is important, especially for the products of lymphoid cells, which are mobile after having picked up the message in the microenvironment throughout the body and therefore their immunoregulatory products, (lymphokines, monokines, interleukins and other cytokines), despite being of local hormone character, may act in fact systemically.
[0069] The central role of cytokines is to control the direction, amplitude, and duration of immune responses and to control the (re)modeling of tissues, be it developmentally programmed, constitutive, or unscheduled. Unscheduled remodeling is that which accompanies inflammation, infection, wounding, and repair. Individual cytokines can have pleiotropic (multiple), overlapping and sometimes contradictory functions depending on their concentration, the cell type they are acting on, and the presence of other cytokines and mediators. Thus the information which an individual cytokine conveys depend on the pattern of regulators to which a cell is exposed, and not on one single cytokine. It is supposed that all cytokines form the specific system or network of communication signals between cells of the immune system, and between the immune system and other organs. In this inter-cell signaling network, the signal is usually transferred by means of a special set of cytokines.
Table 1. Cytokines With Anti-inflammatory Activities
; Cytokines Cellular Sources Major Activities
Monocyte/macrophage Specific inhibitor of IL- lα- and IL-lβ-mediated
IL- Ira dendritic cells cellular activation at the IL-I cellular receptor level
T cells (Th2), mast cells, B Promotes Th2 lymphocyte development; inhibition of
IL-4 cells, stromal cells LPS-induced proinflammatory cytokine synthesis
T cells, B cells, monocytes, Inhibition of TNF and IL-I production by
IL-6 PMNs macrophages
Inhibition of monocyte/macrophage and neutrophil
Monocyte/macrophage, T cytokine production and inhibition of ThI -type
IL-IO cells (Th2), B cells lymphocyte responses
Inhibits proinflammatory cytokine response by monocyte/macrophages and promotes Th2
IL-I l Stromal cells, fibroblasts lymphocyte response
Shares homology with IL-4 and shares IL-4 receptor;
IL- 13 T cells (Th2) attenuation of monocyte/macrophage function
Constitutively expressed in Inhibition of monocyte/macrophage MHC class II
TGF-β many cell lines expression and proinflammatory cytokine synthesis
* PMN = polymorphonuclear cell.
Table 2. Soluble Cytokine Receptors With Anti-inflammatory Activities
Soluble Receptor Cellular Sources Major Activities
Soluble TNF receptor Binds to TNF trimers in the circulation, p55 (sTNFRI or preventing membrane -bound TNF receptor-TNF sTNFRp55) Multiple cell lines ligand interactions
Soluble TNF receptor Binds to TNF trimers in the circulation, p75 (sTNFRII or preventing membrane -bound TNF receptor-TNF sTNFRp75) Multiple cell lines ligand interactions
B cells, neutrophils, Binds to circulating IL- 1 ligands in the plasma,
Soluble IL-I receptor bone marrow preventing IL-IB from binding to the IL-I type 2 (sIL-lRII) precursors receptor type 1
Membrane-bound IL-I B cells, neutrophils, Decoy receptor that lacks intracellular signaling receptor type 2 (mlL- bone marrow function and competes with type 1 IL-IR for IL- IRII) precursors 1 ligand binding at the cell membrane
Soluble extracellular domain of IL- 18 receptor
IL- 18 binding protein Splenocytes, multiple that functions as a decoy receptor and binds (IL- 18BP) other cell lines circulating IL- 18 Detection of Cytokine Biomarkers
[0070] The biomarkers of the invention can be detected in a sample by any means. Methods for detecting the biomarkers are described in detail in the materials and methods and Examples which follow. For example, immunoassays, include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, fluorescent immunoassays and the like. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[0071] Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding an antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4°C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4°C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
[0072] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or n T) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1. [0073] ELISAs comprise preparing antigen (i.e. neural biomarker), coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
[0074] Other preferred methods also include the use of biochips. Preferably the biochips are protein biochips for capture and detection of proteins e.g. cytokines, antibodies etc. Many protein biochips are described in the art. These include, for example, protein biochips produced by Packard BioScience Company (Meriden CT), Zyomyx (Hayward, CA) and Phylos (Lexington, MA). In general, protein biochips comprise a substrate having a surface. A capture reagent or adsorbent is attached to the surface of the substrate. Frequently, the surface comprises a plurality of addressable locations, each of which location has the capture reagent bound there. The capture reagent can be a biological molecule, such as a polypeptide or a nucleic acid, which captures other biomarkers in a specific manner. Alternatively, the capture reagent can be a chromatographic material, such as an anion exchange material or a hydrophilic material. Examples of such protein biochips are described in the following patents or patent applications: U.S. patent 6,225,047 (Hutchens and Yip, "Use of retentate chromatography to generate difference maps," May 1, 2001), International publication WO 99/51773 (Kuimelis and Wagner, "Addressable protein arrays," October 14, 1999), International publication WO 00/04389 (Wagner et al., "Arrays of protein-capture agents and methods of use thereof," July 27, 2000), International publication WO 00/56934 (Englert et al., "Continuous porous matrix arrays," September 28, 2000).
[0075] In general, a sample containing the biomarkers or antibodies to a biomarker, e.g. IL-I, IL-6, IL-10, is placed on the active surface of a biochip for a sufficient time to allow binding. Then, unbound molecules are washed from the surface using a suitable eluant. In general, the more stringent the eluant, the more tightly the proteins must be bound to be retained after the wash. The retained protein biomarkers now can be detected by appropriate means.
[0076] Analytes captured on the surface of a protein biochip can be detected by any method known in the art. This includes, for example, mass spectrometry, fluorescence, surface plasmon resonance, ellipsometry and atomic force microscopy. Mass spectrometry, and particularly SELDI mass spectrometry, is a particularly useful method for detection of the biomarkers of this invention.
[0077] Preferably, a laser desorption time-of-flight mass spectrometer is used in embodiments of the invention. In laser desorption mass spectrometry, a substrate or a probe comprising markers is introduced into an inlet system. The markers are desorbed and ionized into the gas phase by laser from the ionization source. The ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of-flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of markers of specific mass to charge ratio.
[0078] Matrix-assisted laser desorption/ionization mass spectrometry, or MALDI-MS, is a method of mass spectrometry that involves the use of an energy absorbing molecule, frequently called a matrix, for desorbing proteins intact from a probe surface. MALDI is described, for example, in U.S. patent 5,118,937 (Hillenkamp et al.) and U.S. patent 5,045,694 (Beavis and Chait). In MALDI-MS the sample is typically mixed with a matrix material and placed on the surface of an inert probe. Exemplary energy absorbing molecules include cinnamic acid derivatives, sinapinic acid ("SPA"), cyano hydroxy cinnamic acid ("CHCA") and dihydroxybenzoic acid. Other suitable energy absorbing molecules are known to those skilled in this art. The matrix dries, forming crystals that encapsulate the analyte molecules. Then the analyte molecules are detected by laser desorption/ionization mass spectrometry. MALDI-MS is useful for detecting the biomarkers of this invention if the complexity of a sample has been substantially reduced using the preparation methods described above.
[0079] Surface-enhanced laser desorption/ionization mass spectrometry, or SELDI-MS represents an improvement over MALDI for the fractionation and detection of biomolecules, such as proteins, in complex mixtures. SELDI is a method of mass spectrometry in which biomolecules, such as proteins, are captured on the surface of a protein biochip using capture reagents that are bound there. Typically, non-bound molecules are washed from the probe surface before interrogation. SELDI is described, for example, in: United States Patent 5,719,060 ("Method and Apparatus for Desorption and Ionization of Analytes," Hutchens and Yip, February 17, 1998,) United States Patent 6,225,047 ("Use of Retentate Chromatography to Generate Difference Maps," Hutchens and Yip, May 1, 2001) and Weinberger et al., "Time-of-flight mass spectrometry," in Encyclopedia of Analytical Chemistry, R.A. Meyers, ed., pp 11915-11918 John Wiley & Sons Chichesher, 2000.
[0080] Markers on the substrate surface can be desorbed and ionized using gas phase ion spectrometry. Any suitable gas phase ion spectrometers can be used as long as it allows markers on the substrate to be resolved. Preferably, gas phase ion spectrometers allow quantitation of markers.
[0081] In one embodiment, a gas phase ion spectrometer is a mass spectrometer. In a typical mass spectrometer, a substrate or a probe comprising markers on its surface is introduced into an inlet system of the mass spectrometer. The markers are then desorbed by a desorption source such as a laser, fast atom bombardment, high energy plasma, electrospray ionization, thermospray ionization, liquid secondary ion MS, field desorption, etc. The generated desorbed, volatilized species consist of preformed ions or neutrals which are ionized as a direct consequence of the desorption event. Generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The ions exiting the mass analyzer are detected by a detector. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of the presence of markers or other substances will typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of markers bound to the substrate. Any of the components of a mass spectrometer (e.g., a desorption source, a mass analyzer, a detector, etc.) can be combined with other suitable components described herein or others known in the art in embodiments of the invention. [0082] In another embodiment, an immunoassay can be used to detect and analyze markers in a sample. This method comprises: (a) providing an antibody that specifically binds to a marker; (b) contacting a sample with the antibody; and (c) detecting the presence of a complex of the antibody bound to the marker in the sample.
[0083] To prepare an antibody that specifically binds to a marker, purified markers or their nucleic acid sequences can be used. Nucleic acid and amino acid sequences for markers can be obtained by further characterization of these markers. For example, each marker can be peptide mapped with a number of enzymes (e.g., trypsin, V8 protease, etc.). The molecular weights of digestion fragments from each marker can be used to search the databases, such as SwissProt database, for sequences that will match the molecular weights of digestion fragments generated by various enzymes. Using this method, the nucleic acid and amino acid sequences of other markers can be identified if these markers are known proteins in the databases.
[0084] Alternatively, the proteins can be sequenced using protein ladder sequencing. Protein ladders can be generated by, for example, fragmenting the molecules and subjecting fragments to enzymatic digestion or other methods that sequentially remove a single amino acid from the end of the fragment. Methods of preparing protein ladders are described, for example, in International Publication WO 93/24834 (Chait et al.) and United States Patent 5,792,664 (Chait et al.). The ladder is then analyzed by mass spectrometry. The difference in the masses of the ladder fragments identify the amino acid removed from the end of the molecule.
[0085] Any suitable method can be used to detect a marker or markers in a sample. For example, an immunoassay or gas phase ion spectrometry can be used as described above. Using these methods, one or more markers can be detected. Preferably, a sample is tested for the presence of a plurality of markers. Detecting the presence of a plurality of markers, rather than a single marker alone, would provide more information for the diagnostician. Specifically, the detection of a plurality of markers in a sample would increase the percentage of true positive and true negative diagnoses and would decrease the percentage of false positive or false negative diagnoses.
[0086] The detection of the marker or markers is then correlated with a probable diagnosis of neural injury and/or neuronal disorders. In some embodiments, the detection of the mere presence or absence of a marker, without quantifying the amount of marker, is useful and can be correlated with a probable diagnosis of neural injury and/or neuronal disorders. For example, IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-IO, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β, fragments, variants and mutants thereof; can be more frequently detected in patients with neuronal injury than in normal subjects.
[0087] In other embodiments, the detection of markers can involve quantifying the markers to correlate the detection of markers with a probable diagnosis of neural injury, degree of severity of neural injury, diagnosis of neural disorders and the like. Thus, if the amount of the markers detected in a subject being tested is higher compared to a control amount, then the subject being tested has a higher probability of having such injuries and/or neural disorders.
[0088] Similarly, in another embodiment, the detection of markers can further involve quantifying the markers and/or total protein to correlate the detection of markers with a probable diagnosis of neural injury, degree of severity of neural injury, diagnosis of neural disorders and the like, wherein the markers are present in lower quantities in CSF or blood serum samples from patients than in blood serum samples of normal subjects. Thus, if the amount of the markers detected in a subject being tested is lower compared to a control amount, then the subject being tested has a higher probability of having neural injury and/or neural disorder.
[0089] When the markers and/or total protein are quantified, they can be compared to a control. A control can be, e.g. , the average or median amount of marker present in comparable samples of normal subjects in whom neural injury and/or neural disorders, is undetectable. The control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount. For example, if a test sample is obtained from a subject's cerebrospinal fluid and/or blood serum sample and a marker is detected using a particular probe, then a control amount of the marker is preferably determined from a serum sample of a patient using the same probe. It is preferred that the control amount of marker is determined based upon a significant number of samples from normal subjects who do not have neural injury and/or neuronal disorders so that it reflects variations of the marker amounts in that population.
[0090] Data generated by mass spectrometry can then be analyzed by a computer software. The software can comprise code that converts signal from the mass spectrometer into computer readable form. The software also can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a "peak" in the signal corresponding to a marker of this invention, or other useful markers. The software also can include code that executes an algorithm that compares signal from a test sample to a typical signal characteristic of "normal" and human neural injury and determines the closeness of fit between the two signals. The software also can include code indicating which the test sample is closest to, thereby providing a probable diagnosis.
[0091] Antibodies directed against any one of the cytokine biomarkers (e.g., IL-I, IL- lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM- CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β, fragments, variants and mutants thereof) can be used, as taught by the present invention, to detect and diagnose neurological injury. Various histological staining methods, including immunohistochemical staining methods, may also be used effectively according to the teaching of the invention.
[0092] One screening method for determining whether a sample contains, for example, IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β- NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β proteins, peptides or fragments thereof comprises, for example, immunoassays employing radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA) methodologies, based on the production of specific antibodies (monoclonal or polyclonal) to IL-10 protein. Any sample can be used, however, preferred samples comprising the biomarkers are blood, serum, plasma. Venipuncture (blood), urine and other body secretions, such as sweat and tears, can also be used as biological samples. For example, in one form of RIA, the substance under test is mixed with diluted antiserum in the presence of radiolabeled antigen. In this method, the concentration of the test substance is inversely proportional to the amount of labeled antigen bound to the specific antibody and directly related to the amount of free labeled antigen. Other suitable screening methods is readily apparent to those of skill in the art.
[0093] The present invention also relates to methods of detecting cytokine biomarker proteins or fragments thereof, in a sample or subject. For example, antibodies specific for IL- lβ, IL-6, IL-10 proteins, or fragments thereof, may be detectably labeled with any appropriate marker, for example, a radioisotope, an enzyme, a fluorescent label, a paramagnetic label, or a free radical.
[0094] Methods of making and detecting such detectably labeled antibodies or their functional derivatives are well known to those of ordinary skill in the art. The term "antibody" refers both to monoclonal antibodies, which are a substantially homogeneous population and to polyclonal antibodies, which are heterogeneous populations. Polyclonal antibodies are derived from the sera of animals immunized with an antigen. Monoclonal antibodies (MAbs) to specific antigens may be obtained by methods known to those skilled in the art. See, for example, U.S. Pat. No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. It is appreciated that Fab and F(ab')2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of an for example, IL-10 proteins, peptides or fragments thereof, according to the methods disclosed herein in order to detect and diagnose neurological injury or associated diseases thereof in the same manner as an intact antibody. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). [0095] An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody that can also be recognized by that antibody. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
[0096] An "antigen" is a molecule capable of being bound by an antibody that is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies that may be evoked by other antigens. The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the cytokine biomarkers. Thus, the antibodies (or fragments thereof) useful in the present invention may be employed histologically to detect or visualize the presence of IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF- β, proteins, peptides, or fragments thereof.
[0097] Such an assay for detecting biomarkers, typically comprises incubating a biological sample from a subject suspected of having such a condition in the presence of a detectably labeled binding molecule (e.g., antibody) capable of identifying a biomarker and detecting the binding molecule which is bound in a sample. [0098] Thus, in this aspect of the invention, a biological sample may be treated with nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled, with for example, anti-IL-lβ, and/or anti-IL-6 and/or anti-IL-10 specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means. By "solid phase support" is intended any support capable of binding antigen or antibodies. Well-known supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will note many other suitable carriers for binding monoclonal antibody or antigen, or is able to ascertain the same by use of routine experimentation.
[0099] One embodiment for carrying out the diagnostic assay of the present invention on a biological sample containing cytokine biomarkers (e.g. IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β) comprises contacting a detectably labeled antibody specific for a desired biomarker. For illustrative purposes, 11-10 is used as a non-limiting example. A detectably labeled anti- IL-10 specific antibody is bound to a solid support to effect immobilization of anti-IL-10 specific antibody; contacting a sample suspected of containing IL-10 or fragments thereof on the said solid support; incubating the detectably labeled anti-IL-10 specific antibody with the support for a time sufficient to allow the immobilized anti-IL-10 specific antibody to bind to IL-10 and fragments thereof. These steps are followed by washing and detecting the bound label and thereby detecting and quantifying IL-10 and fragments thereof. [0100] Alternatively, labeled anti-IL-10 specific antibody and/or IL-10 protein complexes in a sample may be separated from a reaction mixture by contacting the complex with an immobilized antibody or protein which is specific for an immunoglobulin, e.g., Staphylococcus protein A, Staphylococcus protein G, anti-IgM or anti-IgG antibodies. Such antiimmunoglobulin antibodies may be polyclonal or preferably monoclonal. The solid support may then be washed with a suitable buffer to give an immobilized IL-10/labeled anti- IL-IO specific antibody complex. The label may then be detected to give a measure of IL-10 protein. The specific concentrations of detectably labeled antibody and IL-10, the temperature and time of incubation, as well as other assay conditions may be varied, depending on various factors including the concentration of protein in the sample, the nature of the sample, and the like. The binding activity of a given lot of anti-IL-10 antibody may be determined according to well-known methods. Those skilled in the art is able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation. [0101] One of the ways in which the anti-IL-10 specific antibody can be detectably labeled is by linking the same to an enzyme. This enzyme, in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the anti-IL-10 specific antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β- galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
[0102] Detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling the anti-IL-10 specific antibodies or antibody fragments, it is possible to detect IL-10 protein or fragments thereof, through the use of radioimmunoassays.
[0103] The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are: 3H, 125I, 1311, 35S, 14C, and preferably 125I. [0104] It is also desirable to label the anti-IL-10 specific antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. [0105] The anti-IL-10 specific antibody can also be detectably labeled using fluorescence emitting metals such as ] 2Eu, or others of the lanthanide series. These metals can be attached to the anti-IL-10 specific antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The anti-IL-10 specific antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged anti-IL-10 specific antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
[0106] The anti-IL-10 specific antibody may also be labeled with biotin and then reacted with avidin. Likewise, a bioluminescent compound may be used to label the anti-IL-10 specific antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
[0107] Detection of the anti-IL-10 specific antibody may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material. In the case of an enzyme label, the detection can be accomplished by calorimetric methods that employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. [0108] For the purposes of the present invention, the IL-10 protein and/or fragments thereof, that are detected by this assay may be present in a biological sample. Any sample containing an IL-10 protein or fragments thereof, can be used. However, one of the benefits of the present diagnostic invention is that invasive tissue removal may be avoided. Therefore, preferably, the sample is a biological solution such as, for example, plasma, amniotic fluid, blood, serum, urine and the like. However, the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions that allow the use of other samples. Thus, the diagnosis of neurological injury and/or disease can be established by a simple, non-invasive blood immunoassay that reveals IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β protein levels and/or fragments thereof, greatly increased over normal levels.
[0109] There are many different in vivo labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include radioactive isotopes and paramagnetic isotopes. Those of ordinary skill in the art will know of other suitable labels for binding to the antibodies used in the invention, or is able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the antibodies can be done using standard techniques common to those of ordinary skill in the art.
[0110] An important factor in selecting a radionuclide for in vivo diagnosis is that the half-life of a radionuclide be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host is minimized. Ideally, a radionuclide used for in vivo imaging will lack a particulate emission, but produce a large number of photons in the 140-200 keV range, which maybe readily detected by conventional gamma cameras.
[0111] For in vivo diagnosis radionuclides may be bound to antibody either directly or indirectly by using an intermediary functional group. Intermediary functional groups that are often used in binding radioisotopes that exist as metallic ions to immunoglobulins are DTPA and EDTA. Typical examples of ions that can be bound to immunoglobulins are 99mTc, 123I,
111In, 1311, 97Ru, 67Cu, 67Ga, 125I, 68Ga, 72As, 89Zr, and 201Tl.
[0112] For diagnostic in vivo imaging, the type of detection instrument available is a major factor in selecting a given radionuclide. The radionuclide chosen must have a type of decay that is detectable for a given type of instrument. In general, any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention. For example, PET, gamma, beta, and MRI detectors can be used to visualize diagnostic imagining.
[0113] The antibodies useful in the invention can also be labeled with paramagnetic isotopes for purposes of in vivo diagnosis. Elements that are particularly useful, as in
Magnetic Resonance Imaging (MRI), include 157Gd, 55Mn, 162Dy, and 56Fe.
[0114] The antibodies useful in the present invention are also particularly suited for use in in vitro immunoassays to detect the presence of an IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-
8, IL-10, IL-I l, IL- 12 (p40), IL- 12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-
10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and
TGF-β protein or fragments thereof, in body tissue, fluids (such as CSF, blood, plasma or serum), or cellular extracts. In such immunoassays, the antibodies may be utilized in liquid phase or, preferably, bound to a solid-phase carrier, as described above. [0115] Those of ordinary skill in the art will know of other suitable labels that may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m- maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.
[0116] Removing a histological specimen from a patient, and providing the combination of labeled antibodies of the present invention to such a specimen may accomplish in situ detection. The antibody is preferably provided by applying or by overlaying the labeled antibody to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL- 12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β protein or fragments thereof, but also the distribution of protein on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
[0117] The binding molecules of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested (i.e., blood, plasma or serum) and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
[0118] Typical, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody- antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether antigen is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of antigen.
[0119] In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
[0120] In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays. [0121] The above-described in vitro or in vivo detection methods may be used in the detection and diagnosis of neurological injury without the necessity of removing tissue. Such detection methods may be used to assist in the determination of the stage of neural deterioration in neurological injury and/or disease by evaluating and comparing the concentration of an IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF- β protein or fragments thereof, in the biological sample.
Kits
[0122] In yet another aspect, the invention provides kits for aiding a diagnosis of neural injury, wherein the kits can be used to detect the markers of the present invention. For example, the kits can be used to detect any one or more of the markers described herein, which markers are differentially present in samples of a patient and normal subjects. The kits of the invention have many applications. In another example, the kits can be used to identify compounds that modulate expression of one or more of the markers in in vitro or in vivo animal models to determine the effects of treatment.
[0123] In one embodiment, a kit comprises (a) an antibody that specifically binds to a marker; and (b) a detection reagent. Such kits can be prepared from the materials described above, and the previous discussion regarding the materials (e.g., antibodies, detection reagents, immobilized supports, etc.) is fully applicable to this section and will not be repeated. Optionally, the kit may further comprise pre-fractionation spin columns. In some embodiments, the kit may further comprise instructions for suitable operation parameters in the form of a label or a separate insert.
[0124] In another embodiment, the kit comprises (a) a panel or composition of biomarkers (b) a detecting agent. The panel or composition of biomarkers included in a kit include at least one biomarker and/or a plurality of biomarkers in order to diagnose in vivo location of neural injury. These biomarkers include: IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-I l, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β protein or fragments thereof.
[0125] In another preferred embodiment, the antibodies are specific for at least one biomarker from each neural cell type. The composition of biomarkers is diagnostic of neural injury, damage and/or neural disorders. The antibodies bind to: IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β protein or fragments thereof.
[0126] In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen. [0127] In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface -bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. T he reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.). [0128] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96- well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with bio tiny lated antigen(s).
[0129] Optionally, the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a marker detected in a sample is a diagnostic amount consistent with a diagnosis of neural injury, degree of severity of the injury, subcellular localization, neuronal disorder and/or effect of treatment on the patient.
[0130] In another embodiment, a kit comprises: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent is suitable for binding a marker, and (b) instructions to detect the marker or markers by contacting a sample with the adsorbent and detecting the marker or markers retained by the adsorbent. In some embodiments, the kit may comprise an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluant allows detection of the markers using gas phase ion spectrometry. Such kits can be prepared from the materials described above, and the previous discussion of these materials (e.g., probe substrates, adsorbents, washing solutions, etc.) is fully applicable to this section and will not be repeated. [0131] In another embodiment, the kit may comprise a first substrate comprising an adsorbent thereon (e.g., a particle functionalized with an adsorbent) and a second substrate onto which the first substrate can be positioned to form a probe which is removably insertable into a gas phase ion spectrometer. In other embodiments, the kit may comprise a single substrate which is in the form of a removably insertable probe with adsorbents on the substrate. In yet another embodiment, the kit may further comprise a pre-fractionation spin column (e.g., Cibacron blue agarose column, anti-HSA agarose column, size exclusion column, Q-anion exchange spin column, single stranded DNA column, lectin column, etc.). [0132] Optionally, the kit can further comprise instructions for suitable operational parameters in the form of a label or a separate insert. For example, the kit may have standard instructions informing a consumer how to wash the probe after a sample is contacted on the probe. In another example, the kit may have instructions for pre-fractionating a sample to reduce complexity of proteins in the sample. In another example, the kit may have instructions for automating the fractionation or other processes. [0133] The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification.
[0134] All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.
EXAMPLES
Example 1: Identifying Biomarkers in Patients
[0135] Human Patient population: For TBI patients: Patients > 15 years of age with severe TBI defined as having a sum GCS of < 8 on the post-resuscitation admission neurological examination and requiring a ventriculostomy catheter for clinical management. [0136] For control patients: Patients > 15 years of age requiring an intrathecal catheter or lumbar puncture to rule out meningitis or a subarachnoid bleed or required as part of their routine anesthetic or surgical management (e.g. endovascular aortic aneurysm stent repair, selected orthopedic procedure).
[0137] Cerebrospinal fluid (CSF) samples from TBI patients: CSF will be obtained from all consented subjects in whom a ventriculostomy catheter is placed as part of their standard medical care after TBI. The first CSF sample will be obtained at the time the ventriculostomy catheter is placed. Timed CSF samples (1 ml) will be collected at the time of the insertion of the ventriculostomy catheter (usually this will be within the first 6 hr after injury), and then at 6hr, 12hr, 18hr, 24hr and then every 24hr until the catheter is no longer needed for clinical management up to a maximum of 7 days. Pooled CSF, collected in the drainage bag as a result of CSF removal to control intracranial pressure (ICP) (per routine clinical care), will be collected on the same schedule as the timed CSF samples. The volume of CSF removed will be recorded.
[0138] CSF samples from patients not suffering TBI (control group): Control group samples will be obtained from patients undergoing a lumbar puncture with no evidence of brain injury (e.g., those admitted to the ED to rule out meningitis or a subarachnoid bleed). CSF samples from control subjects will be obtained only at T = 0. In addition, CSF drained through the catheter as part of routine care will be collected from the patient' s collection bag instead of being discarded. Other control samples will be obtained from patients undergoing endovascular aortic aneurysm stent repair. CSF samples will also be obtained from patients who have an either an intrathecal catheter or spinal drain as part of their routine anesthetic or surgical management.
[0139] Blood samples: Blood samples will be collected from TBI or control groups and archived Blood samples will be collected on the same schedule as CSF samples. When ever possible, blood samples will be obtained at the same time blood for routine laboratory studies is obtained. If a patient has no laboratory studies ordered but has vascular access (venous or arterial line in place), the study sample will be drawn from the patient's vascular access catheter. Additionally, blood samples will be collected from the jugular bulb venous catheter (used clinically to monitor brain oxygenation). The total amount of blood volume sampled will approximately be 55 ml over the entire study.
[0140] Cytokine ELISAs: Commercial sandwich ELISA' s for cytokines (e.g. IL-6, IL-10) are used. ELISAs will be performed according to manufacturer's instructions. For example, IL-6 assay kits are from Biosource (#KAC1261 for normal sensitivity, KHC0061 for high sensitivity). A 96-well microplate spectrophotometer (Gemini XS, Molecular Devices) will be used for recording all readings. Signal optimization will be conducted with the protein or glutamate and diluted with normal human CSF fluid, in each case. The standard curve will be run for each independent analysis of human CSF or serum samples. Standard curve fitting always employs a correlation coefficient of 0.95 or larger.
Example 2: Evaluation of rat IL-6 and IL-10 immunological parameters following exposure to methamphetamine and MDMA
[0141] Abuse Club drugs abuse of psychostimulant such as Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA ) abuse represents an immediate problem for the user and a potential long range problem as well. These drugs target a variety of brain cells that are critical to mood, motor and other behaviors. Rather than think that these drug effects are limited to the presence of the drugs in the body and brain, our recent work has have shown that mechanical injury (traumatic brain injury) and drug abuse insults (particularly METH) share common cell injury mechanisms. Since neuron-inflammation is a signature event in TBI, without wishing to be bound by theory, cytokines levels may also be up-regulated in rat brain exposed to both MDMA and METH. In this study we evaluated serum IL-6 and IL-IO concentrations in rats after 24 hrs exposure to 40mg/kg acute regimen of METH and MDMA using ELISA technique. These were compared to rats with traumatic brain injury models and controls. Body weight was measured for the four groups. Interestingly, IL-6 and IL-10 were shown to be elevated in the METH, MDMA, and the TBI groups compared to the saline group suggesting a major role for an inflammatory process involved in these brain insults indicating that drug abuse in fact is a stressor to the immune system. Body weight was shown to drop significantly in the METH group and to a lesser extent in the MDMA groups while it was shown to be relatively stable in TBI and saline groups.
[0142] Acute METH and MDMA model treated animals: All experiments were carried out in male Sprague Dawley rats. Rats were divided into 3 groups each consisting of n =4. Each group received either METH, MDMA at a dosage of 40 mg/kg intraperitoneally. Control group physiological saline (vehicle group). The rats ranged in weight from 250 to 275 g. Animals were 90 days old at the time of sacrifice. All experiments were performed in compliance with NIH guidelines on animal care. After 24 h, animals were briefly anaesthetized and immediately killed by decapitation. Blood was collected and was kept at room temperature for 20 minutes and then centrifuged to collect serum. Serum was kept at - 800C for further use.
[0143] TBI and naive samples serum collection: A controlled cortical impact (CCI) device was used to model TBI in male Sprague-Dawley rats as described elsewhere. TBI injury was performed on 4 animals, each mounted in a stereotactic frame and impacted in the right cortex (ipsilateral) with a 5-mm diameter aluminum impactor tip at a velocity of 3.5 m/sec to a depth of 1.6-mm. Simultaneously, four naϊve control animals were kept under the same environmental conditions, but did not receive an impact injury. Serum collection was performed similar to the METH MDMA Saline groups described else where. All procedures involving animal handling and processing were done in compliance with guidelines set forth by the University of Florida Institutional Animal Care and Use Committee and the National Institutes of Health guidelines. [0144] IL-6, IL-IO ELISA: IL-6 and IL-IO levels were determined by enzyme immunoassay (Bender MedSystems). The procedure described by the manufacturer was followed. Sera from blood collected 20 rat animals divided into the five different groups
(TBI, Naϊve, METH, MDMA and control Saline, each n: 4) were used to establish reference values. The cut off values of the kit is 15.6 pg/ml.
[0145] In our previous in vivo and in vitro studies we have shown a common neurotoxic effect of both TBI and drug abuse (methamphetamine, MDMA) as shown by the degradation of the alpha spectrin neuronal protein (Figures 2A, 2B).
[0146] We utilized a novel approach using IL-6, IL-10 rat ELISA cytokine kit to evaluate the pro-inflammatory pattern of these cytokines, in both TBI and drug abuse models.
Interestingly, IL-6 and IL-10 showed significant increase in both TBI and drug abuse models
Figures IA and IB.
[0147] IL-6 is a pro-inflammatory cytokine secreted by T cells and macrophages to stimulate immune response to trauma, especially burns or other tissue damage leading to inflammation.
[0148] IL-10 is a pleiotropic cytokine that can exert either immunosuppressive or immunostimulatory effects on a variety of cell types. The inhibitory effects of IL-10 on mouse THl cytokine synthesis was found to be indirect and due to the inhibitory effects of mouse IL-10 on the accessory function and antigen-presenting capacity of monocyte/macrophages .
[0149] Robust inflammation was detected in the brain as a result of acute treatment with
40 mg/kg of both methamphetamine and to a lesser extent MDMA. Our data suggests significant brain inflammation in rats treated with 40 mg/kg of Meth that is akin to that observed with a severe brain trauma (1.6 mm controlled cortical impact) (Figures IA and
IB). We also observed a less robust, but statistically significant, increase in cytokine IL-6 at a 40 mg/kg dose of MDMA. The 40 mg/kg dose given to the rats is equivalent to 150 mg/kg of either Meth or MDMA in humans, and would correspond to a typical overdose level.
Example 2: Elevation of pro-inflammatory and anti-inflammatory Cytokines in Rat Serum after Acute Methamphetamine Treatment and Traumatic Brain Injury
[0150] Material and Methods
[0151] Animals: All procedures involving animal handling and processing were done in compliance with guidelines set forth by the University of Florida Institutional Animal Care and Use Committee and the National Institutes of Health guidelines (IACUC). Animals were housed in groups of two per cage and maintained on a 12 h light/dark cycle (lights on 7 AM - 7 PM). Food and water were available ad libitum. All experiments were carried out in male Sprague Dawley rats which were divided into two groups: experimental drug group and a saline vehicle control group consisting of n=4.
[0152] Drug Administration: Pharmacologic agent (+/-) Methamphetamine hydrochloride (8.3mg/ml) (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9% saline. Rats were intraperitoneally (i.p.) injected with METH in a bolus of 0.3 cc to achieve 10 mg/kg dose. This was repeated 4 times every hour to deliver the final dosage of 40 mg/kg. Saline group received similar injection of physiological saline.
[0153] Body Weight Measurement and Serum Collection: Rats were weighed immediately prior to their first injection and then again after 24 hrs prior to sacrifice 24. In case of TBI treated animals, rats were weighed prior to TBI procedure and then after 48 hrs prior to sacrifice. After the desired time periods, rats were briefly anaesthetized with 3-4% isoflurane and were sacrificed by decapitation; blood was collected from the trunk in plain vacutainer tubes. Serum was isolated from collected blood samples by centrifugation (4,000 x g) and stored at -800C until use. Serum was isolated from collected blood samples by centrifugation (4,000 x g) and stored at -800C until use.
[0154] TBI Animal model: A controlled cortical impact (CCI) device was used to model TBI in male Sprague-Dawley rats as described elsewhere (Pike et al., 1998, Neuroreport 9, 2437-42). TBI injury was performed on seven animals, each mounted in a stereotactic frame and impacted in the right cortex (ipsilateral) with a 5 -mm diameter aluminum impactor tip at a velocity of 3.5 m/sec to a depth of 1.6-mm. Seven naϊve control animals were kept under the same environmental conditions, but did not receive an impact injury. Naϊve and injured animals (48 hrs post injury) were sacrificed by decapitation.
[0155] Measurement of Cytokine release using Cytokine Antibody Kit: Acute METH samples, TBI samples and control saline samples were evaluated with the RayBio™ Cytokine Array kit 1.1 (RayBio tech Inc. Nocross, GA) according to the manufacturer's instructions. The cytokine membrane consisted of 19 different secreted cytokine spotted in duplicates along with positive and negative controls. This assay can simultaneously measure cytokines' relative levels with high specificity. Serum obtained from acute METH treated animals, TBI animals and control saline animals were incubated with antibody cytokine membrane to determine the relative concentrations of cytokines. The relative densities of individual spots were measured using ImageJ software for spot analysis. Densitometric readings were analyzed and normalized using RayBio™ Antibody Array Analysis Tool software (RayBiotech Inc. Nocross, GA).
[0156] Densitometry Evaluation: Densitometric quantification of the sample array films was performed using Epson expression 8836XL high-resolution flatbed scanner and NIH ImageJ densitometry software (version 1.6, NIH, Bethesda, MD). The densitometry values were evaluated for statistical significance with SigmaStat software (Version 2.03, Systat Software Inc.). All data presented are expressed as mean ± SEM. Student's t-test was used to draw comparisons between intensities in the treated group (METH and TBI) vs. control (saline) group.
[0157] IL-6 and IL-10 ELISA Procedure: IL-6 and IL-10 levels were determined by an enzyme linked immunoassay purchased from Bender MedSystems (Vienna, Austria) which was applied to validate the protein chip data. Serum cytokine levels were measured from 12 rats (TBI, Naϊve, METH, MDMA and control saline) according to the manufacturer instructions. The cut off value of serum IL-6 and IL-10 levels are 12 pg/ml and 1.47 pg/ml respectively. The values given are quoted in pg/ml. [0158] Results
[0159] Changes in rat weight after acute METH administration and TBI insult: Changes in rat body weight after METH administration are shown in Figure 4. Maximum weight loss was achieved after 24 hrs post METH treatment. It was shown that post METH treatment weight loss reached up to 30 grams (12%) of the rat body weight which may be attributed to the METH induced increased locomotor activity. This significant weight loss was not observed post TBI insult. In addition to this significant weight decrease, METH treated rats exhibited violent behavior shown by increased fighting tendencies. Furthermore, this was accompanied by teeth loss, eye redness and severe sweating. These stereotyped behavior and increased locomotor activity are associated to the METH-induced central release of dopamine and norepinephrin (Ginawi, O. T., et al. (2005). Regul Toxicol Pharmacol 41, 122-7; Ginawi, O. T., et al. (2004). J Physiol Pharmacol 55, 357-69).
[0160] Altered Cytokines Levels via Protein Cytokine Array: Serum analysis of the rat Cytokine Antibody Array revealed a correlation between increasing levels of certain cytokines with the two brain insults (acute METH administration and TBI). It is of interest that TBI and acute METH administration showed elevated levels of the three cytokines IL- lβ, IL-6 and IL-10. These cytokines elevation in TBI insult is indicative of immunostimulatory activation which is consistent with TBI human data reflecting a simultaneous elevation of different pro-inflammatory and anti-inflammatory cytokines after traumatic brain injury in both CSF and serum. These findings are suggestive that acute METH treatment involves an inflammatory process in addition to the classical METH- mediated neurotoxic events. The biological significance of the altered cytokines is to be discussed late.
[0161] IL-6 and IL-10 Levels Quantitation by ELISA : In order to check the reliability of the relative differences of cytokines observed by using the Cytokine Antibody Array, we quantified IL-6 and IL-10 cytokines; two cytokines that showed upregulation in both TBI and acute METH insult by a conventional ELISA, as described above. It was shown that similar to the Cytokine Antibody Array, ELISA yielded a significant statistical elevation of IL-6 and IL-10 after both acute METH insult (IL-6=1608 pg/ml and IL-I= 944 pg/ml) and TBI insult (IL-6 =1742 and IL-10=759 pg/ml) when compared to saline control (IL-6=1608 pg/ml and IL-I= 944 pg/ml) as shown in Figures 4A and 4B.
[0162] Discussion: Based on the accumulated data that traumatic brain injury and acute METH abuse share a number of biochemical commonalities such as the dual activation of the caspase and calpain systems and since the activation of immune response is a signature feature in TBI in response to injury of neuronal cells. Thus, we hypothesized that immunomodulatory response may be involved also in acute METH abuse. [0163] In this study, we evaluated differential serum cytokine expression in acute METH treatment (40 mg/ml) using cytokine antibody array kit that detects 19 different cytokines which was compared to that of TBI and control saline serum as shown in Figures 5A-5B and 6A-6B. Interestingly, our cytokine data revealed an elevation in serum IL- lβ, IL-6 and IL-10 in both TBI and acute METH treatment. IL-6 and IL-10 levels which were quantified by sandwich ELISA confirmed the cytokine antibody array data as shown in Figures 6A-6B. [0164] Our TBI data showing elevated IL-6 and IL-10 are in concert with human TBI studies which show a marked elevation of both pro-inflammatory and anti-inflammatory cytokines including IL-I β, IL-6, IL-8, TNF-α and IL-10 which are detected at different time points in time course studies. Cytokine differential changes are detected in serum or CSF and are related to BBB disruption.
[0165] IL-6, it is a pleiotropic cytokine that regulates immune response mechanism and is produced not only by the immune cells but epithelial cells; IL- lβ is a pro-inflammatory cytokine produced by macrophages and dendritic cells. Serum elevation of these two cytokines may be indicative of the pro -inflammatory environment at the injured area which is infiltrated by peripheral immune cells due to BBB leakage. [0166] IL-IO was shown to be elevated after TBI; IL-IO is an anti-inflammatory cytokine that inhibits several macrophages functions including pro-inflammatory cytokine production. However, the paradoxical presence of elevated IL-10 along with pro-inflammatory cytokines, IL- lβ and IL-6, can be related to the time dependant expression of these cytokines. In our current study, our data revealed that acute METH treatment exhibited similar cytokine elevation (IL- lβ, IL-6 and IL-10) to that of TBI as shown in Figures 6A-6B and Figures 7A- 7B. This was accompanied by behavioral finding of significant weight loss (Figure 4) along with teeth loss, increased fighting tendencies and excessive sweating which can be related to increased locomotor activity. Taken together, these data indicate that inflammatory response activation may be involved in the METH induced neurotoxicity. Use of anti- inflammatory agents aimed at suppressing cytokine production and/or the activation of different inflammatory cells such as macrophages and lymphocytes from producing them presents a novel strategy in treating METH induced toxicity.
[0167] Finally, our data showed an elevation of IL-10. Although generally considered an immunosuppressive molecule; IL-10 exhibits some immunostimulatory properties as shown in different studies. In this context, several studies have shown that IL-10 enhances the function of natural killer cells which lead to antigen presentation by antigen presenting cells. Whether IL-10 is acting as an immuno- suppressive agent attenuating the inflammatory actions of IL- lβ and IL-6 cytokines or as a stimulatory to the immune response, its serum elevation would be of similar pattern detected in the TBI insult so as to quell the damage of the potent secreted pro-inflammatory cytokines.
[0168] In conclusion, our cytokine data are indicative of an immunomodulation process occurring post acute METH exposure and TBI brain insults. The dual elevation of the proinflammatory cytokines IL- lβ and IL-6 indicate a potentially harmful neuron inflammation response to neuronal cell death in METH abuse or TBI. Future work would necessitate the evaluation of cytokines levels at different time points for a better understanding of immune system dynamics involvement in the area of drug abuse and TBI. Finally, the finding that pro-inflammatory cytokines are present after acute METH abuse indicate a possible use of different anti-inflammatory agents; this indeed, presents a novel strategy in treating METH induced toxicity.
[0169] Data obtained from methamphetamine exposure show and validate our hypothesis that acute methamphetamine injury, a self inflicted chemical brain injury, is similar to brain injury. Immunological and biochemical evidence indicates that traumatic brain injury and acute methamphetamine share a number of cell death mechanisms in the central nervous system and this in turn may necessitate the use a common treatment represented by antiinflammatory drugs.
Other Embodiments
[0170] While the above specification contains many specifics, these should not be construed as limitations on the scope of the invention, but rather as examples of preferred embodiments thereof. Many other variations are possible.

Claims

What is claimed is:
1. A method of diagnosing neurological injury in a patient comprising identifying cytokines and cytokine levels in a sample from an injured patient, wherein the cytokines detected are elevated as compared to normal individuals.
2. The method of claim 1, wherein the cytokines detected in a sample comprise: IL- 1, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-IO, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β- NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β.
3. The method of claim 1, wherein the cytokines detected comprise IL-lβ, IL-6 and IL-10.
4. The method of claim 2, wherein the sample is selected from the group consisting of saliva, sputum, blood, blood plasma, serum, urine, tissue, cells, and liver.
5. The method of claim 1, wherein the cytokine levels are compared to known biomarkers of neurological injury.
6. A method of treating a patient suffering from neurological disorders comprising measuring the cytokine levels in the patients and treating the patient with anti-inflammatory agents.
7. The method of claim 6, wherein a patient is treated with agents that decrease cytokine levels identified as diagnostic of neurological damage as compared to a normal individual.
8. The method of claim 6, wherein decreasing the cytokine levels decreases neurological injury.
9. The method of claim 6, wherein the cytokines in a sample from a patient comprise: IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-IO, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP- 1, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β.
10. The method of claim 9, wherein the cytokines comprise IL- lβ, IL-6 and IL-10.
11. The method of claim 9, wherein the sample is selected from the group consisting of cerebrospinal fluid, blood, blood plasma, serum, urine, tissue, cells, and organs.
12. A method of monitoring effectiveness of treatment of neural injury comprising measuring cytokine levels and/or total protein in a biological sample obtained from said subject, wherein detected levels of cytokines and/or total protein compared to normal subjects is indicative of the effectiveness of treatment of neurological injury.
13. The method of claim 12, wherein decreased levels of cytokines and/or total protein in a biological sample obtained from said subject, as compared to levels detected prior to treatment in the same patient, is prognostic of the effectiveness of treatment.
14. The method of claim 12, wherein the cytokines comprise: IL-I, IL- lβ, IL-2, IL- 4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β.
15. The method of claim 12, wherein the cytokines comprise IL-lβ, IL-6 and IL-10.
16. The method of claim 12, wherein the sample is selected from the group consisting of cerebrospinal fluid, blood, blood plasma, serum, urine, tissue, cells, and organs.
17. The method of claim 12, wherein cytokine levels and/or total protein are detected using an immunoassay.
18. The method of claim 17, wherein the immunoassay is an ELISA.
19. The method of claim 12, wherein cytokine levels and/or total protein are detected using a biochip array.
20. The method of claim 19, wherein the biochip array is a protein chip array.
21. The method of claim 19, wherein the biochip array is a nucleic acid array.
22. The method of claim 19, wherein the surface of the biochip array comprises one or more antibodies specific for cytokines.
23. The method of claim 19, wherein the surface of the biochip array comprises single or double stranded nucleic acids.
24. The method of claim 19, wherein the surface of the biochip array comprises proteins, peptides or fragments thereof.
25. The method of claim 19, wherein the surface of the biochip array comprises amino acid probes.
26. The method of claim 19, wherein the surface of the biochip array comprises phage display libraries.
27. The method of claim 19, wherein the one or more cytokines and/or total protein are immobilized on the biochip array.
28. The method of claim 19, wherein immobilized one or more cytokines and/or total protein are subjected to laser ionization to detect the molecular weight of the markers.
29. A method of identifying the severity of neurological injury course in a patient, comprising measuring cytokines and/or total protein in a biological sample obtained from said subject, wherein an elevated level of cytokines and/or total protein compared to normal subjects is indicative of the severity of neurological injury.
30. The method of claim 29, wherein the cytokines comprise: IL-I, IL-lβ, IL-2, IL- 4, IL-5, IL-6, IL-8, IL-10, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenin, GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β.
31. The method of claim 30, wherein the cytokines comprise IL-lβ, IL-6 and IL-10.
32. The method of claim 29, wherein one or more cytokines and/or total protein are detected using an immunoassay.
33. The method of claim 32, wherein the immunoassay is an ELISA.
34. The method of claim 29, wherein cytokines and/or total protein are detected using a biochip array.
35. A kit comprising: IL-I, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-IO, IL-Il, IL-12 (p40), IL-12 (p70), IL-13, Angiogenic GM-CSF, IFN-γ, MCP-I, IP-10, MIP-Ib, MIP-3α, TNF-α, VEGF, TIMP-I, β-NGF, CINC-2, Leptin, MCP-I, LIX and TGF-β, peptides, fragments or antibodies thereof.
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