Title:

Blood assessment of injury

Document Type and Number:

United States Patent Application 20070059745

Kind Code:

Abstract:

Methods of injury assessment in an individual include the steps of determining a pattern of expression exhibited by blood cells obtained from an individual and comparing the pattern of expression exhibited by the obtained blood cells to an injury database to assess the injury.

Representative Image:

Inventors:

Sharp, Frank R. (Cincinnati, OH, US)
Tang, Yang (Cincinnati, OH, US)
Lu, Aigang (Cincinnati, OH, US)

Plaque It!

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 of U.S. Provisional Application Ser. No. 60/253,568 filed Nov. 28, 2000.

FIELD OF THE INVENTION

The present invention is directed toward methods of assessing injury in an individual, wherein injury is defined as cell death, cell dysfunction, or genetic abnormalities either acquired or inherent, any of which are present in an occult, acute or chronic stage. More particularly, the invention is directed toward methods of injury assessment which comprise determining a pattern of expression exhibited by obtained blood cells and comparing the pattern of expression exhibited by the obtained blood cells to an injury database to assess the injury.

BACKGROUND OF THE INVENTION

Non-invasive diagnostic methods such as computed tomography (CT) and magnetic resonance imaging (MRI) are useful in diagnosing injury resulting from ischemia, tumors, bleeding, trauma, toxins, infection, autoimmune disease and other etiologies. Invasive imaging methods include positron emission tomography (PET) and single photon emission computed tomography (SPECT), which require the injection of radioisotopes, and cerebral angiography and myelography, which require the injection of radiopaque dyes. A further invasive procedure for assessing injury is through the use of a biopsy.

Individuals who are admitted into medical facilities often have altered states of consciousness associated with cellular death or dysfunction, which may be caused by many factors, including cardiac arrest, strokes, hemorrhages, hypoglycemia episodes, head injuries, seizures, psychiatric diseases, infection, toxins, drugs, as well as coma due to liver, renal, endocrine or pulmonary failure. Such patients may be unable to respond to requests regarding a medical history or conditions. Further, it is often difficult to transport or to use imaging technology on artificially ventilated patients in intensive care units or post-surgical units. Still further, it is complicated to perform a biopsy when the source or the cause of the injury may be unknown. Thus, it would be useful to have a convenient method of assessing injuries that does not require a biopsy, imaging or transfer of the patient, and can be done with procedures no more invasive than the withdrawal of a blood sample.

Neither CT nor MRI are useful for diagnosing injury where there is isolated dysfunction or isolated loss of neurons or individual cells in the blood, brain, spinal cord, lung, muscles, nerves or other organs. For example, there are no convenient methods for determining whether injury to cells in the brain, blood, muscle, nerves, heart, lung, endocrine glands or other organs has occurred following hypoglycemia, hypoxia, drug over-dose, coma, status epilepticus, stroke, or severe hypotension due to cardiac arrest or other causes. In addition, even with these imaging methods there are numerous injuries that cannot be conveniently or adequately assessed. For example, patients suffering cardiac arrest with cardiovascular collapse often have diffuse neuronal injury in the brain and in other organs that cannot be visualized. Similarly, injury caused by hypoxia, hypoglycemia, or status epilepticus cannot be diagnosed with such methods. Thus, it would be useful to have a convenient and adequate method to assess injury states.

Many individuals remain asymptomatic for an injury for numerous years. Such individuals do not seek medical treatment because the injury is not prevalent. In addition, such individuals cannot report an accurate medical history because they are not aware of a hidden medical condition. Therefore, it is nearly impossible to accurately assess injury in these individuals when symptoms are not overtly expressed. Thus, it would be useful to have a convenient method of assessing asymptomatic injuries to continuously monitor an individual's health.

The prior art teaches that specific genes or proteins have been identified that correspond with a particular specific disease. In addition, these genes and proteins can be classified using microarray technology. The identification and measurement of these specific genes and proteins allow a specific disease to be diagnosed.

For Example, Barone, et al., J. Cereb. Blood Flow Metab., 19(8):819-834 (1999), teach that transforming growth factor (TGF), tissue necrosis factor (TNF), interleukin-1 (IL-1), interleukin-8 (IL-8), heat shock proteins, and metalloproteinases may be induced, for example, in the brain during a stroke. Bergeron et al., European Journal of Neuroscience, 11:4159-4170 (1999), teach that hypoxia-inducible factor-1 (HIF-1), glucose transporter-1 (GLUT-1), and several glycolytic enzymes are upregulated in, for example, the brain during focal ischemia. HIF-1 is induced by hypoxia, but not by hypoglycemia—making this gene a candidate for distinguishing between hypoxia and hypoglycemia in blood, the brain and other organs. Sharp et al., TINS, 22:97-99 (1999), teach that heat shock proteins (HSPs) and glucose-regulated proteins (GRPS) are produced in response to ischemia and other stresses. HSPs are induced in response to denatured proteins, GRPs are induced in response to low glucose, and ORPs (oxygen regulated proteins) are induced in response to low oxygen. Martens et al., Stroke, 29:2363-2366 (1998), teach that S-100 protein, a calcium-binding protein, may be a serum marker of brain damage useful for clinical assessment. Martens et al. further teach that cardiac arrest may produce cerebral damage that can be detected by release of neuron-specific enolase to the cerebrospinal fluid and eventually to the blood.

Microarrays of DNA have been used to classify types of cancer, as taught by Alizadeh et al., Nature, 403:503-511 (2000), and Golub et al., Science, 283:531-537 (1999). Microarrays have also been used in analyzing inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, as taught by Heller et al., Proc. Natl. Acad. Sci., U.S.A., 94:2150-2155 (1997). Friend et al, (Rosetta Inpharmactics, Inc.) U.S. Pat. No. 6,218,122 (2001), teach a method for monitoring disease states and levels of effect of therapies using gene expression profiles derived from cellular constituents indicating aspects of the biological state of the cell, such as RNA or protein abundances or activity levels. Erlander et al (Ortho-McNeil Pharmaceutical, Inc.) WO 00/28092 (2000), teach a method for the production of gene expression profiles from a selected set of cells residing in a given tissue/organ. Friend et al, (Rosetta Inpharmactics, Inc.) WO 00/24936 (2000), teach methods of using co-regulated genesets to enhance the detection and classification of specific gene expression patterns for a specific biological state. Ralph et al., (Urocor, Inc.) U.S. Pat. No. 6,190,857 (2001), teach that a specific human disease state may be detected in circulating leukocytes by identifying specific genomic markers for the specific disease state.

However, even with the progression in the art, there remains a substantial need for convenient and adequate methods that can assess an injury for an individual. It would also be advantageous to provide methods of assessment which could be conveniently and adequately used in particular individuals who are asymptomatic, artificially ventilated and/or in altered states of consciousness, and that go beyond current methods of clinical diagnosis.

There is also a substantial need for methods of assessment that could utilize a relatively non-invasive procedure for diagnosis, prognosis, and/or monitoring an injury state.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide convenient methods of assessing injury.

In accordance with one aspect of the invention, there are provided methods of injury assessment in an individual. The methods comprise the steps of determining a pattern of expression exhibited by blood cells obtained from the individual and comparing the pattern of expression exhibited by the blood cells to an injury database to assess the injury. In specific embodiments, the pattern of expression may be a pattern of gene expression, protein expression, or combinations thereof, and the injury database may be a genomic database, proteomic database, or combinations thereof. Furthermore, the injury database may be based on a specific organ or a specific injury cause or disease.

In accordance with another aspect of the invention, there are provided methods of stroke injury assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess stroke injury.

In accordance with yet another aspect of the invention, there are provided methods of hypoxia injury assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury databases to assess hypoxia injury.

In accordance with a further aspect of the invention, there are provided methods of hypoglycemia injury assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury bank to assess hypoglycemia injury.

In accordance with yet another aspect of the invention, there are provided methods of seizure injury assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess seizure injury.

In accordance with yet another aspect of the invention, there are provided methods of movement disorder injury assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess movement disorder injury.

In accordance with yet another aspect of the invention, there are provided methods of diabetes injury assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess diabetes injury.

In accordance with yet another aspect of the invention, there are provided methods of infectious disease assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess infectious disease injury.

In accordance with yet another aspect of the invention, there are provided methods of immune mediated disease assessment of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess immune mediated disease injury.

In accordance with yet another aspect of the invention, there are provided methods of efficacy or toxicity assessment, or combinations thereof, of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess efficacy or toxicity, or combinations thereof. The methods can be used, for example, for assessing efficacy and/or toxicity of drugs or environmental toxins.

In accordance with yet another aspect of the invention, there are provided methods of psychosis assessment, or combinations thereof, of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess psychosis.

In accordance with yet another aspect of the invention, there are provided methods of headache assessment, or combinations thereof, of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess headache.

In accordance with yet another aspect of the invention, there are provided methods of genetic disorder assessment, or combinations thereof, of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess the genetic disorder.

In accordance with yet another aspect of the invention, there are provided methods of proliferative disease assessment, or combinations thereof, of an individual comprising the steps of obtaining a peripheral blood sample from the individual, capturing a pattern of expression, defining a pattern of expression, and comparing the pattern of expression exhibited by the blood cells to an injury database to assess the proliferative disease disorder.

The present methods are advantageous in providing convenient, relatively non-invasive diagnosis of injury in occult, acute or chronic stages. Additional embodiments, objects and advantages of the invention will become more fully apparent in view of the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description will be more fully understood in view of the drawings in which:

FIG. 1 a is a Venn diagram showing the numbers of genes that were upregulated more than twofold in blood 24 hours after brain ischemia (BI), brain hemorrhage (BH), and sham surgery (S), compared with untouched control individuals, as described in Example 2;

FIG. 1 b is a Venn diagram showing the numbers of genes that were downregulated more than twofold in blood 24 hours after kainate (K), insulin-glucose (IG), and hypoxia (H), compared with untouched control individuals, as described in Example 2;

FIG. 2 is a cluster analysis of the pattern of expression obtained from individuals with kainate, insulin-glucose, hypoxia, brain ischemia, brain hemorrhage, as compared to sham surgery and untouched control individuals, as described in the Example 2;

FIG. 3 a is a graph which demonstrates the identification of Dead Box Y Isoform, which is differentially expressed in two groups of patients, males and females, as described in Example 3;

FIG. 3 b is a graph which demonstrates the identification of Ribosomal Protein S4 Y Isoform, which is differentially expressed in two groups of patients, males and females, as described in Example 3;

FIG. 4 is a graph which demonstrates that genes SEQ ID NO:1 and SEQ ID NO:2 are expressed more highly in Parkinson's individuals as compared to other individuals without Parkinson's, as described in Example 4;

FIG. 5 is a cluster analysis of the expression obtained from pediatric epilepsy patients prior to being treated compared to the expression of these individuals after being treated with anticonvulsant valporate (VPA) or the anticonvulsant carbamazepine (CPZ), as described in the Example 8;

FIG. 6 is a cluster analysis of the pattern of expression obtained from individuals with neurofibromatosis, as described in Example 9;

FIG. 7 is a cluster analysis of the pattern of expression obtained from individuals with bipolar, as described in Example 10;

FIG. 8 is a cluster analysis of the pattern of expression obtained from individuals with acute migraine headaches, as described in Example 11;

FIG. 9 is a cluster analysis of the pattern of expression obtained from individuals with schizophrenia, as described in the Example 12; and

FIG. 10 is a cluster analysis of the pattern of expression obtained from individuals with Tourettes, as described in the Example 13.

DETAILED DESCRIPTION

Upon injury, the blood, in particular the blood cells, will be exposed to environmental stresses, immune responses or additional effects associated with the injury. The inventors have found that blood cell responses can be used to determine whether there has been injury to neurons or injury to other cells in the body, the cause of the injury, and/or the degree of the injury. Methods in accordance with the invention may be used to detect remote injury. In addition, methods in accordance with the invention may be used to assess injury that cannot be conveniently or adequately evaluated by current blood tests, by imaging or biopsy, and may conveniently be used on all individuals, including individuals who are asymptomatic, in altered states of consciousness, and/or who are artificially ventilated. Advantageously, methods in accordance with the present invention are relatively non-invasive and do not require biopsy or the injection of radioisotopes or radiopaque dyes.

As used herein, “assessment” is intended to refer to the prognosis, diagnosis, or monitoring of an injury based upon a pattern of expression from a blood sample. As used herein, “individual”, is intended to refer to an animal, including but not limited to humans, mammals, and rodents. As used herein “blood cells”, is intended to refer to nucleated cells of the blood, including but not limited to red blood cells, white blood cells, lymphocytes, leukocytes, monocytes, macrophages, eosinophils, basophils, polymorphonucleic cells, all other subsets of cells containing RNA or protein, or combinations thereof.

As used herein, “injury” is intended to refer to genetic abnormalities, either inherent or acquired; death of cells; or dysfunction of cells produced by a wide variety of overt or covert states including, but not limited to, diffuse systemic disease, hyperproliferative cellular conditions, including benign, and non-benign or metastatic cancer, hemorrhage, infarction, ischemia, hypoxia, seizures, psychiatric illnesses, neurological diseases, hypoglycemia, trauma, toxins, drugs, organs, inflammatory diseases, autoimmune diseases, infectious diseases, demyelinating diseases, tumors, cancer, endocrine diseases, degenerative and metabolic diseases, including Alzheimer's, and infection, present in an occult, acute or chronic stage.

Autoimmune diseases include, but are not limited to, Graves, Rheumatoid arthritis, Thyroiditis/hypothyroidism, Vitiligo, IDDM, Multiple sclerosis, Primary glomerulonephritis, Systemic lupus erythematosus, Sjogren's, Addison's disease, autoimmune hemolytic anemia, chronic active hepatitis, Goodpasture's syndrome, idiopathic thrombocytopenia purpura, myasthenia gravis, myocarditis, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, relapsing polychondritis, rheumatic fever, scleroderma, and uveitis. Psychiatric illnesses include, but are not limited to, schizophrenia, generalized anixiety, panic disorders, post traumatic stress, obsessive compulsive, phobias, social anxiety disorder, major depressive disorder, bipolar, alchol and drug abuse, and eating disorders.

As used herein, “organ injury” is meant to refer to injury to one or more organs, including but not limited to, the following: brain, organs of the special senses including eyes, ears and nose, the central nervous system, the spinal cord, nerves, muscles, heart, lung, kidney, liver, genitalia, endocrine glands, bladder, gastrointestinal system, joints, bones, blood vessels, and blood cells, including red blood cells and white blood cells, and including lymphocytes, leukocytes, monocytes, macrophages, eosinophils, basophils, and all other cells found in blood.

As used herein, “glucose-inducible genes” is intended to refer to genes which are induced by changes in serum or blood glucose levels, usually low glucose levels, and decreased with high glucose levels; while “glucose-related proteins” is intended to refer to gene products which are produced or which levels are varied in response to changes in serum or blood glucose levels, preferably low glucose levels. “Low glucose levels” is intended to refer to glucose levels below the range generally regarded by physicians as normal. As used herein, “hypoxia-induced factors” is intended to refer to factors which are produced or which levels are varied in response to hypoxia.

As used herein, a “genomic injury bank” refers to a library composed of DNA, RNA, or combinations thereof, isolated from blood samples. As used herein, a “proteomic injury bank” refers to a library composed of protein isolated from blood samples. As used herein, an “injury database” refers to a database comprising a pattern of expression or patterns of expressions indicative of a single or different states of injury, including but not limited to pattern of gene expression, protein expression, or combinations thereof. The injury database may be based on a specific organ or a specific injury cause or disease. Organ specific injury databases include, but are not limited to, brain injury database, spinal cord injury database, blood injury database, muscle injury database, nerve injury database, lung injury database, liver injury database, heart injury database, kidney injury database, genitalia injury database, eye injury database, ear injury database, nose injury database, teeth injury database, bone injury database, white blood cell injury database, endocrine gland injury database, gastrointestinal injury database, blood vessel injury database, or combinations thereof. Cause/disease specific injury databases include, but are not limited to, global ischemic injury database, focal ischemic profile, status epilepticus injury database, hypoxia injury database, hypoglycemia injury database, cerebral hemorrhage injury database, hemorrhage injury database for one or more organs, diabetes complications injury database, psychosis injury database, psychiatric disease injury database, bipolar injury database, schizophrenia injury database, headache injury database, acute migraine headache, database, endocrine disease injury database, uremia injury database, injury database for ammonemia with hepatic failure, toxin overdose injury database, drug overdose injury database, Alzheimer's disease injury database, Parkinson's disease injury database, Tourettes disease injury database, muscle disease injury database, proliferative disease injury database, neurofibromatosis injury database, nerve disease injury database, other dementing illness injury database, inflammatory diseases injury database, autoimmune diseases. injury database, infectious diseases injury database, demyelinating diseases injury database, trauma injury database, tumors injury database, cancer injury database, degenerative and metabolic diseases including Alzheimer's injury database, genetic or familial diseases injury database, or combinations thereof.

As used herein “stroke” or “cerebrovascular accident” is intended to refer to cerebral infarction resulting from lack of blood flow and insufficient oxygen to the brain. As used herein, “infarction” is intended to refer to tissue/cell death. In an ischemic stroke, the blood supply is cut off due to a blockage in a blood vessel, while in a hemorrhagic stroke the blood supply is cut off due to the bursting of a blood vessel.

As used herein, “pattern of expression” is meant to refer to the representation of molecules, including but not limited to genes, proteins or combinations thereof, in an injury state, which are upregulated, downregulated or embody no change. As used herein, “expression method” is meant to refer to any method known in the art that can define a pattern of expression, such as the significance analysis of microarrays and class prediction, as taught by Tusher, Proceedings National Academy of Sciences, 98: 5116 (2001). These methods may assess injury at a point minutes, hours, days or weeks after the injury has occurred, owing to rapid and/or prolonged expression of the molecules indicating the injury.

Patterns of expression may be derived from, but are not limited to, the following detailed injuries. For example, in individuals who sustain a brief period of severe hypoglycemia (low serum glucose) because of oral or injected hypoglycemics or because of severe illnesses there may be an induction of glucose-inducible genes in all of the blood cells, including polymorphonuclear cells (neutrophils), lymphocytes and macrophages. Hypoglycemia may also damage brain cells, blood cells, cells in the pancreas, cells in the heart, lung and other organs. Thus, gene and protein expression in the blood cells may change in response to the hypoglycemia.

In individuals who sustain a period of pure hypoxia during anesthesia or while on a respirator there may be an induction of a set of genes specific for hypoxia; therefore, glucose-inducible genes may not be induced. In contrast, in individuals sustaining a cardiac arrest, wherein the brain, other organs and blood become ischemic for a length of time, there may be an induction of genes regulated by low glucose and low oxygen, as well as genes that are related to acidosis and ischemia. Thus, the genomic and/or proteomic response which may be observed in blood cells during episodes of pure hypoxia may differ from those observed in blood cells during episodes of pure hypoglycemia.

An individual having status epilepticus has brain injury manifested by isolated neuronal injury. The removal of such dead neurons is performed by monocytes and macrophages. Thus, during status epilepticus there may be selective change in genomic and/or proteomic expression of macrophages. Further, during repeated seizures there may be little white cell hypoxia or hypoglycemia, thus, hypoxia-induced factors, glucose-related proteins and heat shock proteins will not be induced. Additionally, during prolonged seizures there may be massive sympathetic discharge. The individuals may have elevation of catecholamines (e.g., epinephrine) that may stimulate adrenergic receptors in the blood cells.

If a individual is suffering from one or several focal strokes, blood cells respond to the site of the injury, the brain, and the response is targeted to brain antigens with removal and repair of neurons, glia, and vessels. During severe ischemic hypotension and infarction of the brain or other organs, hypoxia-induced factors, glucose-related proteins, and heat shock proteins are all induced. In heavy metal toxicity, heat shock proteins may be induced.

It has been found that molecules regulate in accordance with an injury state to determine a pattern of expression. In an embodiment of the invention, the number of molecules necessary to define a pattern of expression is at lease about 10. In an embodiment of the invention, the number of molecules necessary to define a pattern of expression is at lease about 50. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is at least about 200. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is at least about 500. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is at least about 1000. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is at least about 5000. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is about at least 10,000. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is about at least 50,000. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is about at least 100,000. In a further embodiment of the invention, the number of molecules necessary to define a pattern of expression is all molecules represented in the injury state. The upper and/or lower limit of molecules necessary to define a pattern of expression may similarly vary in individuals applications of the present method, and in specific embodiments may be 10, 50, 200, 500, 1000, 5000, 10,000, 100,000, or the like.

In accordance with another embodiment of the invention, the molecules, which may be used in determining a pattern of expression by blood cells include, but are not limited to, intermediate metabolism, immune-related molecules, cytokines, chemokines, immediate early genes, structural genes, neurotransmitters, receptors, signaling molecules, oncogenes and proto-oncogenes, heat shock and stress genes, transporters, trophic and growth factors, cell cycle genes, lipid metabolism, arachidonic acid metabolism, free radicals and free radical scavengers, metal binding, transporting genes, or combinations thereof.

In accordance with yet another embodiment of the invention, various enzymes whose expression may be evaluated comprise aldolase-A, lactase, dehydrogenase-A, phosphofructokinase-L, pyruvate kinase-M, hypoxia-inducible factor, or combinations thereof, while heat shock proteins whose gene expression may be evaluated comprise ubiquitin, HSP10, HSP27, HSP25, HSP32 (also known as heme oxygenase-1 or HO-1), HSP47, HSP60, HSC70 (also known as HSC73), HSP70 (also known as HSP72), HS90, HS100/105, or combinations thereof.

In accordance with a further embodiment of the invention, the classes of genes and proteins further comprise intermediate-early genes (IEGs), the genes for hypoxia-inducible factor 1 (HIF-1), glucose transporter-1 (GLUT-1), glycolytic enzymes, transforming growth factor (TGF), tissue necrosis factor (TNF), interleukin-1 (IL-1), interleukin-1 receptor antagonist (IL-1 RA), interleukin-8 (IL-8), heat shock proteins (HSPs), glucose-regulated proteins (GRPs), oxygen-regulated proteins, metalloproteinases, nitric oxide synthase (NOS), cyclooxygenases (COX), poly(ADP-ribose) polymerase (PARP), calcium-binding proteins such as S-100 proteins, histamine H2-receptor, c-jun leucine zipper interactive protein, Glut3, the vesicular monoamine transporter, TNF intracellular domain interacting protein, vascular tyrosine phosphatase, glucose-induced genes, hypoxia-induced genes, transcription factors, signaling factors, growth factors, transmitters, receptors, membrane protein genes, peptides, cytokines, chemokines, structural genes, cell cycle genes, apoptosis-related genes, acidosis-induced genes, ischemia-induced genes, enzymes, kinases, phosphatases, trophic factors, nuclear factors, hormones, or combinations thereof. Hypoxia-induced genes comprise genes for heat shock proteins, genes for nitric oxide synthase, genes for matrix metalloproteinases, genes for cyclooxygenases, genes for growth factors, genes for hypoxia-induced factors such as HIF-1, and genes involved in the production of cytokines, chemokines, adhesion molecules, or combinations thereof. Glucose-induced genes comprise glucose regulated proteins, glycolytic enzymes, glycosylated proteins, genes as listed in Table 3, or combinations thereof. Acidosis-induced genes comprise the genes as listed in Table 2, genes listed in Table 3, or combinations thereof. Ischemia-induced genes comprise the genes as listed in Table 3 or combinations thereof. Parkinson-related genes may comprise SEQ ID NO:1, SEQ ID NO:2, or combinations thereof.

The pattern of expression exhibited by the obtained blood cells may be captured by any method known to the art. An exemplary method is through the use of microarrays, for example using DNA microarrays, protein microarrays, peptide microarrays, or combinations thereof. Microarrays refer to surface microarrays, membrane microarrays, bead microarrays, solution microarrays, and the like comprised of nucleic acids, nucleic acid mimetics, discrete nucleotide sequences, preferably DNA or RNA sequences, discrete proteins, antibodies, protein fragments, antibody fragments, antibody-mimetics, peptides, peptide-mimetics, organic molecules and/or other molecules capable of selectively and specifically binding specific RNA, DNA or proteins; or subsets of RNA, DNA or protein molecules thus permitting the detection and measurement of the associated molecules for the purpose of capturing a pattern of expression.

In one embodiment of the invention, microarrays are used to capture the pattern of gene expression. The nucleotide sequences in two DNA samples or two RNA samples, such as, for example, the RNA isolated from two different cell populations, are compared by first labeling the samples, mixing the samples and hybridizing them to arrayed DNA spots. Generally each nucleotide sequence is labeled with a different flourescent dye or other labeling technique. As the samples are differentially labeled, it is possible to determine the pattern of gene expression.

To prepare RNA for use in a microarray assay, it is generally purified from total cellular content. Suitable methods of RNA isolation are known in the art and include the use of standard isolation methods, specific columns, or other collection methods. The RNA may be reversed transcribed to complementary DNA (cDNA) and in some applications to complementary RNA (cRNA). Either the labeled cDNA or the labeled cRNA may be used in the microarray assay.

Generally, the cDNA or cRNA samples are labeled, for example, with fluorescent dyes (fluors). Common fluors include Cy3 and Cy5. The labeled samples are referred to as probes. The probes are hybridized to a DNA sequence in the microarray. If the labeled probe contains a cDNA or cRNA whose sequence is complementary to the DNA at a given spot in the microarray, the labeled probe will hybridize to that spot, where it can be detected by its fluorescence. Since the probes are tagged with fluorescent molecules like Cy3 and Cy5 that emit detectable light when stimulated by a laser, the probes may be scanned and the emitted light recorded. The probe may be applied to a microarray, DNA, RNA or protein.

In a further embodiment of the invention, a microarray comprises from about 1,000 to about 100,000 DNA sequences. A sample is obtained from the patient's blood cells and is labeled with a first label, and a second RNA sample which serves as a control is labeled with a second label. The first label and the second label have different emission wavelengths. The labels may be fluors, biotinylated markers or other suitable markers. The labeled patient sample and the labeled control samples are mixed and hybridized to the microarray, or they are hybridized to separate arrays. Generally the microarray is then rinsed to remove any non-hybridized samples. The light emitted from the fluors may be measured using any method known in the art, such as commercially available scanners. The relative abundance of the patient and control samples hybridized to the various DNA sequences of the microarray are determined and a pattern is captured.

In yet another embodiment of the invention, the RNA is isolated from the blood of the hypoglycemia, hypoxia, status epilepticus, ischemic stroke, hemorrhagic stroke, and controls. The RNA is purified using standard methods, and then transcribed either into labeled cDNA or into labeled cRNA. These samples are then applied to custom microarrays that are fabricated using the methods for suppressive subtraction hybridization, or custom arrays made from commercially available cDNA libraries. The experimental samples are labeled with Cy3 and the untouched control or sham control samples are labeled with Cy5. The two samples are mixed and applied to a cDNA array produced from all available rat cDNAs, or from an array produced from cDNAs obtained from the suppressive subtractive hybridization. Altematively, the samples could be applied to currently available commercial arrays from Incyte, Affymetrix, Research Genetics, and other commercial vendors. Alternatively, samples could be applied to proteomic/protein microarrays.

After a pattern of expression has been captured and defined, an injury database can be established for the injury state. Once an injury database is established for the injury state, only one fluorescent dye is necessary to capture the pattern of expression for subsequent samples as the pattern will be compared to the established injury database.

An example of a commercially available microarray is an Affymetrix chip. These arrays are fabricated using spatially patterned, light-directed combinatorial chemical synthesis, and contain hundreds of thousands of oligonucleotides immobilized on the glass surface of the arrays (Affymetrix, Santa Clara, Calif.). For most sequences or EST there are 16 probe 20 mer oligonucleotide pairs, of which 8 a perfect match and 8 are a mismatch where one nucleotide is changed in the middle of the sequence. Each array also contains a number of reference sequences, which after standards are added allows normalization and quantification of the data. The human U95A array is used, having 13000 sequences and EST's.

In an embodiment of the invention, the expression levels of the molecules, captured on the microarray, are ranked from the lowest expressed molecule being assigned a rank of 1 to the most highly expressed molecule. For example, if 100,000 molecules were assessed from a single blood sample, the lowest expressed molecule would be assigned a value of 1 and the most highly expressed molecule a value of 100,000 with every other molecule having a value in between. The ranks of the molecules of individuals with a specific injury or on a specific medication are compared to other individuals with other conditions or to normal healthy controls.

In a further embodiment of the invention, the determination of a pattern of expression further comprises ranking the genes of the captured pattern of expression. The expression levels of the genes, captured on the microarray, are ranked from the lowest expressed gene being assigned a rank of 1 to the most highly expressed gene. For example, if 100,000 genes were assessed from a single blood sample, the lowest expressed gene would be assigned a value of 1 and the most highly expressed gene a value of 100,000 with every other gene having a value in between. The ranks of the genes of individuals with a specific injury or on a specific medication are compared to other individuals with other conditions or to normal healthy controls.

In one embodiment of the invention, microarrays are used to capture the pattern of protein expression. The protein is isolated from either whole blood and/or from white blood cells isolated from whole blood. The protein is then applied to a protein microarray. A protein microarray may be composed of antibodies to all known proteins, antibodies to selected protein subsets, or proteins themselves.

In yet another embodiment of the invention, protein detection is used. Protein detection may include multiple mass spectrophotometric analyses performed in parallel or any other method of detecting hundreds to thousands of proteins at one time from a single blood sample from a single patient. The proteins and antibodies are detected using mass spectrophotometric, fluorescent, radioactive or other techniques and the expression levels of each protein assessed in a manner analogous to detection of multiple RNA species on current oligonucleotide and cDNA microarrays.

In yet another embodiment of the invention, the determination of a pattern of expression further comprises ranking the proteins of the captured pattern of expression. The expression levels of the proteins, captured on the microarray, are ranked from the lowest expressed protein being assigned a rank of 1 to the most highly expressed protein. For example, if 100,000 proteins were assessed from a single blood sample, the lowest expressed protein would be assigned a value of 1 and the most highly expressed protein a value of 100,000 with every other protein having a value in between. The ranks of the proteins with individuals with a specific injury or on a specific medication are compared to other individuals with other conditions or to normal healthy controls.

Any expression method known in the art may be used to define the pattern of expression captured. A preferred method is the Significance Analysis of Microarrays (SAM) and class prediction, as taught by Tusher, Proceedings National Academy of Sciences, 98: 5116 (2001); Golub et al., Science, 286: 531-537(1999). Other expression methods are available, including neural network modeling, clustering, computer programs, and entropy methods, and could be used as alternatives.

The significance analysis of microarray (SAM) and class prediction may be used to define the pattern of expression captured. The significance analysis of microarrays uses permutations of repeated measurements to estimate the percentage of genes or proteins identified by chance. Once the molecules are identified that are regulated in a specific injury, this set of molecules is said to define the pattern expression for that injury. To determine whether an unknown sample is consistent with the normal pattern of expression or is consistent with the pattern for a specific injury, the following general procedure is followed. The expression value for each molecule in the unknown sample is compared to the expression value in the normal set of molecules and in the injury set of genes or proteins. A class prediction method is then used to determine whether the unknown sample fits the normal or injury pattern. To do this, the expression value for each molecule is determined to be closer to the control or the injury state, and a weighted vote is made for each molecule for the injury pattern. The diagnosis of the injury is made if PS>0.3 when PS is the prediction strength, defined as PS=(Vw−V _L)/(Vw+V _L). If there is no difference between the samples, then PS will equal zero and the sample would fall in the class of the control or healthy blood sample. If PS>0.3, then the sample would be classified as the injury state.

In one embodiment of the invention, the most regulated genes or proteins for a given condition that had the lowest variance may be identified using SAM analysis for various medical, neurological, genetic and other conditions. These regulated genes or proteins may be used to define a pattern for each condition, a class prediction, that would be used to analyze unknown samples to determine whether they would fit the pattern for a specific disease or condition or not with a 90, 95 or 99% confidence level.

Once the pattern of expression is captured and defined, the pattern of expression exhibited by the obtained blood cells is compared to an injury database to assess the injury. This database may comprise a pattern of expression or multiple patterns of expression based on a specific organ, a specific injury cause or disease, or combinations thereof. Further, the database may be a commercially available database or a database created from the pattern of expression captured and defined by the obtained blood cells.

In one embodiment of the invention, injury databases for hypoxia, status epilepticus and hypoglycemia, are prepared using blood cell samples. These databases are used to assess the injury of an individual based on the comparison between the pattern of expression of the individual and pattern of expression of the database.

The embodiments, as set forth above, can be used for any injury as the blood expression will differ with each and every different injury and the database will remain constant.

EXAMPLES

In the examples and throughout the present specification, parts and percentages are by weight unless otherwise indicated.

Example 1

This example demonstrates the use of the claimed invention to assess hypoxia, status epilepticus, hypoglycemia, ischemic stroke, and hemorrhagic stroke in individuals. One day after hypoxia, status epilepticus, hypoglycemia, ischemic stroke, and hemorrhagic stroke are produced in adult rats, RNA or protein is isolated from the blood cells and from the brains of these animals. Suppressive-subtractive hybridization is performed on the isolated RNA or protein. The clones, obtained from the suppressive-subtractive hybridization, or the isolated RNA or protein are sequenced. The pattern of genes or proteins expressed in the blood cells following each of these types of injury—hypoxia, status epilepticus, hypoglycemia, ischemic stroke, and hemorrhagic stroke is captured. The pattern of gene or protein expression is defined using an expression method, which then forms a genomic or proteomic organ injury database, which is used in assessing injury in the individuals.

More specifically, adult Sprague Dawley rats (300-350 gm males) are housed in a fully AAALAC accredited Animal Research Facility. All animals are examined upon receipt and any animals with symptoms of disease or other problems are sacrificed. Animals are fed ad libitum, with fresh food and water provided several times weekly. Cages are cleaned on a regular schedule.

A custom hypoxia chamber is constructed comprising four identical chambers wherein inlet and outlet air is controlled and monitored. Any oxygen concentration (0-100%, by volume) can be achieved using computer controlled valves and pumps. The inlet and outlet oxygen concentration in each chamber is measured continuously, as is carbon dioxide, temperature and humidity. The oxygen concentrations can be ramped up or down over any period of time (seconds to hours). Generally, the 8%, by volume, oxygen concentration is ramped down over 30 minutes, and the animals remain at 8% oxygen for 6 hours, after which the oxygen is ramped back up to 21%.

Status epilepticus is produced by intraperitoneally injecting a glutamate analogue/excitotoxin, kainic acid (10 mg/kg i.p.). Animals with kainate-induced seizures are observed following drug administration to ensure that they continue to have complex seizures over a 30 minute period. Animals with seizures longer than 30 minutes and that have neuronal injury demonstrated histologically are included in the study. Animals injected with kainic acid have diffuse neuronal injury 24 hours later.

Regular insulin (20 U sq) is used to induce systemic hypoglycemia. The animals are injected subcutaneously with 10 U regular insulin and go into a coma for several hours. The severe hypoglycemia causes severe diffuse neuronal injury. Animals remain hypoglycemic for a period of 4 hours. The hypoglycemia is then reversed with repeated injections of 25% dextrose (25 cc) given every half hour for two hours as needed. Prolonged hypoglycemia is required to produce neuronal injury in the brain and other organs. These periods of hypoglycemia induce glucose-regulated protein 75 (GRP75) and other glucose regulated proteins in brain and other organs such as the liver and other tissues.

Ischemic stroke is produced by anesthetizing adult rats with isoflurane. A ventral neck incision is made, and the common carotid artery is isolated. The external carotid artery is ligated, and a 4-0 nylon suture advanced into the external carotid artery and then up the internal carotid artery to the bifurcation of the middle and anterior cerebral arteries. The suture is left in place for two hours to produce an infarction (stroke) in the distribution of the middle cerebral artery. Control animals for the stroke are called “sham” animals. These animals are anesthetized, have the neck incision performed, and arteries isolated, but do not have the suture inserted into the artery and do not have an ischemic stroke.

Hemorrhagic stroke is produced by anesthetizing adult rats with isoflurane. The scalp is incised and a burr hole drilled 0.5 mm anterior and 4 mm lateral to bregma. A 25 gauge needle was used to deliver 50 μl of lysed arterial blood 4 mm into the right striatum. The hemorrhage results in cell death around the margins of the hemorrhage.

Untouched, control animals are not injected or touched prior to the experiment. These animals remain awake, do not undergo surgery, but are housed and treated like the other animals described above.

All animals are allowed to survive for 24 hours following each treatment. At that time they are deeply anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) given intraperitoneally. Once anesthetized, the chest is opened and a direct cardiac puncture performed with a syringe and 10 cc of blood is aspirated. Immediately following removal of the blood, the animal is decapitated while deeply anesthetized and the brain removed.

The blood from the animals from the hypoxia group is pooled, as is blood from the animals from the status epilepticus group, the animals from the hemorrhagic stroke group, the animal from the ischemic stroke group, and the animals from the hypoglycemia group. The blood from the untouched control and the sham-operated control animals is pooled as well. White blood cells are separated on a FICOLL® gradient, and the RNA from each pooled group is extracted with Trizol reagent. Subtractive hybridizations are then performed using commercially available kits (ClonTech) to obtain several separate subtraction libraries: control versus hypoxia blood; control versus status epilepticus blood; control versus hypoglycemic blood; control versus ischemic stroke blood; and control versus hemorrhagic stroke blood. Generally there are about 500 to about 1000 clones for each subtraction.

Suppressive subtractive hybridization (SSH) is based on a form of PCR that permits exponential amplification of cDNAs that differ in abundance, whereas amplification of RNAs of similar abundance in the control and experimental populations is suppressed. Alternatively, Representational Difference Analysis (RDA) may be used for performing library subtractions.

Poly A+ RNA from the control bloods (“driver” or “control”) and the hypoxic, hypoglycemic, ischemic stroke, hemorrhagic stroke, or status epilepticus bloods (“tester” or “experimental”) is made, and then quantified on a formaldehyde gel. Each sample is concentrated to a range of from about 1 to about 2 μg/ml. Double stranded (ds) cDNAs are prepared from the two poly A+ RNA samples by reverse transcription. Second strand cDNA synthesis is then performed and the ds cDNAs are digested with a four-base cutting enzyme (Rsa I) that yields blunt ends. The cut ds cDNAs are digested with a four-base cutting enzyme (Rsa I) that yields blunt ends. The cut ds cDNAs are analyzed on a 1%, by weight, agarose gel.

Following this, the tester ds cDNA pool is divided into two equal portions, and the ds cDNA in one portion is ligated with adaptor 1 and the cDNA in the other portion is ligated with adaptor 2 using T4 DNA ligase. Since the ends of the adaptors do not have a phosphate group, only one strand of each adaptor attaches to the 5′ ends of the cDNA. Importantly, the two adaptors (1 and 2R) share a stretch of common sequences that allows them to anneal with each other during PCR. Following successful ligation of the adaptors, hybridization is performed with excess “driver” added to each “tester” sample. The samples are heat denatured and allowed to anneal. The concentration of high and low abundance cDNAs are equalized in the adaptor-ligated population of cDNAs. The cDNAs are equalized due to second-order hybridization kinetics for these differently expressed cDNAs (ClonTech). There is exponential amplification of rare cDNAs in the “tester” samples. During the second hybridization, the two “tester” samples ligated with adaptor 1 and 2R, and the freshly denatured “driver” sample are mixed without denaturing. Only the equalized and subtracted single stranded (ss) tester molecules can re-associate and form double stranded hybrids. The ends (site of different adaptors) are then filled in and these new hybrids are amplified by PCR. Molecules missing the primer annealing sites (adaptor 1 and 2R) cannot be amplified due to suppression of PCR.

The subtracted library is ligated into the T/A cloning vector (Invitrogen, Inc.) and electroporated into phage-resistant bacterial cells (DH10B), which are then stored in glycerol at −80° C. An aliquot (100 μl) of the library is plated on a LB agar plate with the appropriate antibody for the purpose of determining the titer of the library. The T/A cloning vector has a B-galactosidase site that provides the mechanism for color (blue vs white) selection of bacterial colonies that contain a subtracted clone. Positive colonies are inoculated in 96-well plates with antibiotic and 10% glycerol and stored at −80° C. This becomes the original copy of the library. Several controls are performed to help ensure that the procedure worked properly. First, from about 60 to about 80 randomly selected clones are examined on 2% agarose gels to show that the inserts are of the appropriate sizes ranging from about 0.3 to about 1 kb, and that they are of differing sizes and therefore unique. PCR for G3PDH (gyceraldhyde-3-phosphate dehydrogenase) is performed on the subtracted and unsubtracted libraries to ensure that the ubiquitously expressed and unregulated G3PDH is not expressed in the subtracted library.

Clones that show a two fold or greater induction by hypoxia, hypoglycemia ischemic stroke, hemorrhagic stroke, or status epilepticus in the five subtracted libraries are sequenced and compared to currently available rat sequences (GeneBank). The cloned sequences are also subjected to BLAST (basic local alignment search tool, GenBank database) to determine if they match the sequences of known genes. BLAST is a computer program used to search databases to determine if a sequence is similar to that of known or previously cloned genes.

Once a sufficient number of clones are sequenced and their identity determined, genes are selected for further study based upon their expression with each type of injury. For example, glucose regulated genes are induced with hypoglycemia and not with hypoxia and status epilepticus. Hypoxia-inducible factor and its hypoxia-inducible target genes are induced with hypoxia and not with hypoglycemia or status epilepticus. Catecholamine-related genes, like alpha-adrenergic and beta adrenergic-receptors, are induced to a greater extent following status epilepticus as compared to hypoxia or hypoglycemia. Once candidate clones are identified, then the clones are used to perform Northern blots on RNA from bloods of the hypoxic, hypoglycemic, status epilepticus, ischemic stroke, hemorrhagic stroke and control groups. Alternatively, PCR is performed on each sample and the PCR products sequenced to confirm gene induction for each group. Each clone is then used to produce a spot on a microarray.

Northern blots are performed to confirm the specificity of the clones for each gene and to quantify RNA induction. After isolation of RNA, it is incubated with DNase (5 U/ml; Promega) and RNAsin (200 U/ml; Promega) at 37° C. for 30 min. The RNA is ethanol precipitated, dissolved in water and the OD260/280 determined. Four micrograms of RNA are electrophoresed in a 1.5% agarose gel containing 1×MOPS and 7% paraformaldehyde and transferred to a nylon membrane (Nytran, Sleicher and Schuell, Keene, N. H.) for a period of from about 12 to about 18 hours. The RNA is cross-linked to the membrane with UV light at 254 nm (Stratalinker, Stratagene, Calif.). The membrane is stained with 0.02% methylene blue and the position of the 18S and 28S bands marked on the membrane. It is then pre-hybridized at 42° C. for about 1 hour with a mixture of 6×SSC, 0.1% SDS, 10× Denhardt's reagent and 50 μg/ml heat denatured salmon sperm DNA. Clones are labeled using TdT (Gibco BRL) with ³²P-dATP (DuPont-NEN Research Products) and membranes are hybridized at 37° C. overnight in 6×SSC, 1% SDS and 1-4×10 ⁶cpm/ml of the labeled probe. After hybridization, the membranes are washed to a maximum stringency of 6×SSC and 0.1% SDS (sodium dodecyl sulfate) at 55° C. The membranes are then covered with Kodak SB5 autoradiographic film for a period of from about 4 to about 12 hours and developed in Kodak GBX developer. Blots are quantified using an MCID (St. Catherine's, Ontario, Canada) image analysis system.

The fabricated microarray is used to capture the pattern of expression in the injury states of hypoxia, status epilepticus, hypoglycemia, ischemic stroke, and hemorrhagic stroke. An expression method defines the pattern of expression and the pattern of expression is compared to an injury database to assess the injury.

Example 2

This example demonstrates the use of the claimed invention to assess hypoxia, status epilepticus, hypoglycemia, ischemic stroke, and hemorrhagic stroke. One day after hypoxia, status epilepticus, hypoglycemia, ischemic stroke, and hemorrhagic stroke are produced in adult rats, RNA or protein is isolated from the blood cells and from the brains of the animals described in Example 1. The pattern of genes or proteins expressed in the blood cells following each of these types of injury—hypoxia, status epilepticus, hypoglycemia, ischemic stroke, and hemorrhagic stroke is captured on a commercially available microarray (Affymetrix chip). The pattern of gene or protein expression is defined using an expression method, which then forms a genomic or proteomic organ injury database, which is used in assessing injury.

The data below demonstrates the pattern of gene expression in the blood cells and in the brain following specific pathological insults using genomic profiles based on commercially available microarrays. The data demonstrate how a pattern of gene expression is derived, and that the patterns of gene expression for the different pathological states are different from each other. The tables give lists of genes induced in blood and in the brain of animals exposed to hypoxia, stroke, and status epilepticus as compared with untouched control or sham operated control animals. As shown in FIGS. 1 a and 1 b, many genes upregulated or downregulated by each experimental condition were modulated in two or more groups. FIG. 2 presents a cluster analysis of the pattern of expression obtained from individuals with kainate, insulin-glucose, hypoxia, brain ischemia, brain hemorrhage, as compared to sham surgery and untouched control individuals.

For the tables of genes induced in the blood, the genome expression of blood in the hypoxic animals (3 animals) was compared to the genome expression of blood in untouched control animals (3 animals). The genome expression of blood in the animals with status epilepticus (3 animals) was compared to the genome expression of blood in the untouched control animals (3 animals). The genome expression of blood in the animals with stroke (3 animals) was compared to the genome expression of blood in the sham operated control animals (3 animals). In each case the accession number of the gene and the fold change in gene expression is given—with a maximum estimate and a minimum estimate.

Tables 1 to 4 set forth lists of genes induced in the blood in the different conditions. Tables 5 and 6 set forth lists of genes induced in the brain in the different conditions. Note that the genes induced in the blood are different from the genes induced in the brain. Therefore, different organs express different genes. In addition, the genes induced by hypoxia in the blood are different from the genes induced by hypoxia in the brain. That is, the same stimulus induces different genes in different organs. Lastly, even though similar genes are induced in the brain by ischemia (stroke) and kainic acid-induced seizures, there are many differences in the gene expression between the two. Therefore, the pattern of gene expression in the brains of ischemic animals is distinctive from the pattern of expression of the kainate animals, and this pattern can be used to diagnose the different conditions of stroke and status epilepticus, even though many of the same genes are induced in the two conditions.

Table 1 sets forth genes induced in the blood of rats 24 hours following 6 hours of 8% hypoxia (n=3 rats) as compared with genes expressed in the blood of untouched control rats (n=3 rats). The accession number of the gene is given, the name of the gene is given where known, the average fold induction is given, as well as the minimum fold induction is given for each gene. A number of the genes are ESTs that have not yet been subjected to a BLAST search. This list represents the number of genes induced on arrays that contained 8000 genes.

TABLE 1


Accession No.	Name	Average	Minimum


X62950mRNA_f_at	pBUS30 with repetitive elements	10	4.8
rc_AA891933_at		9	1.9
X06827_at	porphobilinogen deaminase	7.1	4.1
rc_AA894273_at		6.1	2.7
X63675_at	Pim-1	6	1.8
D13978_s_at	argininosuccinate lyase	5.1	1.9
X62325cds_r_at	T cell receptor V-alpha J-alpha	5	1.8
rc_AA891737_at		5	1.6
rc_AA891920_at		4.9	2.7
S65555_g_at	gamma-glutamylcysteine synthetase light chain	4.5	2.1
rc_AI233261_i_at		4.4	1.5
X06827_g_at	porphobilinogen deaminase	4.3	2
rc_AA800745_at		4.3	1.5
X17053mRNA_s_at	Rat immediate-early serum-responsive JE gene	4.2	3.9
rc_H33723_at		4.1	2.6
S65555_at	gamma-glutamylcysteine synthetase light chain	4.1	1.9
U39875_at	EF-hand Ca2+-binding protein p22	4	1.8
rc_AI059042_at		4	1.7
M91234_f_at	VL30 element	3.9	2.4
U73030_at	pituitary tumor transforming gene (PTTG)	3.9	1.6
Y13275_at	D6.1A protein	3.9	1.5
M59936cds_at	connexin-31	3.8	2.1
rc_AA852046_s_at		3.8	2.1
rc_AA852046_s_at		3.8	2.1
rc_AI145680_s_at		3.8	1.6
rc_AI045315_f_at		3.8	1.4
M15474cds_s_at	alpha-tropomyosin gene	3.7	2.4
AF102552_s_at	270 kDa ankyrin G isoform	3.7	1.7
M91235_f_at	VL30 element	3.6	2.4
U07201_at	asparagine synthetase	3.5	2
AB015194_at	50 kD glycoprotein (Rh50)	3.5	1.8
U25650_f_at	low affinity nerve growth factor receptor precursor	3.5	1.4
	(LNGFR)
X17053cds_s_at	Rat immediate-early serum-responsive JE gene	3.4	2.1
Y00350_at	uroporphyrinogen decarboxylase	3.4	1.9
rc_AA891880_g_at		3.4	1.3
rc_AI235890_s_at		3.3	2.5
rc_AI235890_s_at		3.3	2.3
AB000199_at	cca2	3.3	1.3
M62388_at	ubiquitin conjugating-protein	3.2	1.8
X89225cds_s_at	L-like neutral amino acid transport activity protein	3.2	1.5
rc_AA858607_at		3.2	1.4
X82396_at	cathepsin B	3.1	2.3
X62660mRNA_g_at	glutathione transferase subunit 8	3.1	1.3
M60666_s_at	alpha-tropomyosin 2	3	1.7
rc_AA926149_g_at		3	1.6
AF076856_s_at	small espin	2.9	1.8
rc_AA892897_at		2.8	1.7
D90401_g_at	dihydrolipoamide succinyltransferase	2.8	1.4
M34134_s_at	brain alpha-tropomyosin (TMBr-2)	2.8	1.4
rc_AA799680_at		2.8	1.4
rc_AI029920_s_at		2.8	1.3
rc_AA891107_at		2.7	1.6
rc_AI235585_s_at		2.7	1.6
X67948_at	channel integral membrane protein 28	2.7	1.6
AF067790_s_at	palmitoyl-protein thioesterase	2.7	1.4
M89945mRNA_g_at	Rat farnesyl diphosphate synthase gene	2.7	1.4
rc_AA819793_at		2.6	1.8
J02592_s_at	glutathione S-transferase Y-b subunit	2.6	1.6
rc_AA893590_at		2.6	1.6
AF090113_g_at	AMPA receptor binding protein	2.6	1.4
M89945mRNA_at	Rat farnesyl diphosphate synthase gene	2.5	1.6
rc_AI180442_at		2.5	1.5
D63774_at	keratin 14	2.5	1.3
rc_AA818025_at		2.4	1.3
rc_AI014094_at		2.4	1.3
D86215_at	brain mRNA for NADH-ubiquinone oxidoreductase	2.3	2.1
rc_AA874827_at		2.3	1.6
rc_AA946368_at		2.3	1.6
U82623_g_at	cytocentrin	2.3	1.6
X12554cds_s_at	heart cytochrome c oxidase subunit VIa	2.3	1.4
AJ009698_g_at	embigin protein	2.3	1.3
D10026_s_at	glutathione S-transferase	2.2	1.7
rc_AA851403_g_at		2.2	1.5
U67138_at	PSD-95/SAP90-associated protein-2	2.2	1.4
D38036_at	Truncated TSH receptor	2.2	1.3
rc_AA892805_g_at		2.2	1.3
rc_AI013513_at		2.2	1.3
rc_AA851887_s_at		2.1	1.6
D13120_s_at	ATP synthase subunit d	2.1	1.4
rc_AA892888_at		2.1	1.4
U82623_at	cytocentrin	2.1	1.4
D16478_at	mitochondrial long-chain enoyl-CoA hydratase	2.1	1.3
rc_AA799612_at		2.1	1.3
AF029240_at	MHC class Ib RT1.S3	2	1.4
J05022_at	peptidylarginine deiminase	2	1.4
rc_AI231472_s_at		2	1.4
rc_AA866477_at		2	1.3
rc_AA875107_at		2	1.3
rc_AI105050_at		2	1.3
rc_AA925752_at		2	1.1
AF050663UTR#1_at	norvegicus activity and neurotransmitter-induced	1.9	1.5
	early gene
X53363cds_s_at	calreticulin	1.9	1.5
S78154_at	inwardly rectifying ATP-regulated K+ channel	1.9	1.4
U24489_at	tenascin-X	1.9	1.4
X63722cds_s_at	vascular cell adhesion molecule-1(VCAM-1)	1.9	1.4
D13212_s_at	N-methyl-D-aspartate receptor subunit (NMDAR2C)	1.9	1.3
D78308_g_at	calreticulin	1.9	1.3
AF017437_g_at	integrin-associated protein form 4 (IAP)	1.8	1.5
X03369_s_at	beta-tubulin T beta 15	1.8	1.5
D45254_g_at	cellular nucleic acid binding protein (CNBP)	1.8	1.4
rc_AI146195_at		1.8	1.4
AF020618_at	progression elevated gene 3 protein	1.8	1.3
AF060174_at	synaptic vesicle protein 2C (SV2C)	1.8	1.3
D10587_at	85 kDa sialoglycoprotein (LGP85)	1.8	1.3
rc_AA799887_s_at		1.8	1.3
rc_AA859957_at		1.8	1.3
X80395cds_s_at	rVAT gene	1.8	1.3
rc_AA892260_at		1.7	1.4
AF017437_at	integrin-associated protein form 4 (IAP)	1.7	1.3
AF073839_s_at	bithoraxoid-like protein	1.7	1.3
Rc_AI169631_s_at		1.7	1.3
U36444cds#1_at	PCTAIRE-1 protein kinase	1.7	1.3
L38437_at	NADH ubiquinone oxidoreductase subunit (IP13)	1.6	1.3
	gene
rc_AI112237_at		1.6	1.3
rc_AA893690_g_at		1.5	1.3

Table 2 sets forth genes induced in the blood of rats 24 hours following kainate induced seizures (n=3 rats) as compared with genes expressed in the blood of untouched control rats (n=3 rats). The accession number of the gene is given, the name of the gene is given where known, the average fold induction is given, as well as the minimum fold induction is given for each gene. A number of the genes are ESTs that have not yet been subjected to a BLAST search. This list was shortened to show only those genes induced at least 2.8 fold. Over 100 genes were induced following kainate on arrays that contained over 8000 genes.

TABLE 2


Accession No.	Name	Average	Minimum


D84485_at	PMSG-induced ovarian mRNA	11.4	3.1
M96159_at	adenylyl cyclase type V	10	2.9
Rc_AA955182_g_at		9	2.3
AF045464_s_at		6.5	2.5
X76697_at	B7 antigen	5.7	2.5
D89863_g_at (M-ras)	M-Ras	5.6	2.3
U66566_at	receptor type protein tyrosine phophatase psi	5.5	4.3
L81138exon	Rps2r gene	5.5	2.3
AF079162_at	patched (ptc)	5.4	3.2
Rc_AA894273_at		5.2	2.8
Rc_AA799614_at		4.7	2.5
AF102552_s_at	ankyrin G isoform	4.6	2.4
M91234_f_at	VL30 element	4.4	2.5
L42855_at	RNA polymerase II transcription factor SIII p18	4.32	3.4
	subunit
Rc_AA852046_s_at		4.3	2.5
AF027571_s_at	phospholipase C-beta 4 isoform (PLC-b4)	4.15	2.5
Rc_AI104924_f_at		4.1	3.3
U73030_at		4.1	2.4
Rc_AA925529_at		4	3
Rc_AA891828_at		4	2.6
M91235_f_at	VL30 element	3.9	3
L81136cds_f_at	Rps2r1 preliminary DNA	3.9	2.7
X06827_at	porphobilinogen deaminase	3.6	3
X60675_at	interleukin 10	3.6	2.3
Z28351exon_s_at	25-hydroxyvitamin D3 24-hydroxylase	3.5	2.3
AF091563_i_at	isolate QIL-LD1 olfactory receptor	3.4	2.4
rc_AI102562_at		3	2.4
S54212_at	ciliary neurotrophic factor receptor alpha	2.8	2.6

Table 3 sets forth genes induced in the blood of rats 24 hours following a stroke produced by filament occlusion of the middle cerebral artery (n=3 rats) as compared with genes expressed in the blood of sham operated control rats (n=3 rats). The accession number of the gene is given, the name of the gene is given where known, the average fold induction is given, as well as the minimum fold induction is given for each gene. A number of the genes are ESTs that have not yet been subjected to a BLAST search. This list was produced from arrays that contained over 8000 genes.

TABLE 3


Accession No.	Name	Average	Minimum


X52196cds_at	five-lipoxyenase activating protein (FLAP)	9.5	1.7
rc_AA866444_s_at		8.8	2.6
Rc_AA892851_at		5.6	3.9
rc_H31722		5.4	2
L18948_at	intracellular calcium-binding protein (MRP14)	4.1	1.7
rc_AA849036		4	2.5
rc_AI043796_s_at		3.9	2.4
D89093_at	cGMP-binding cGMP-specific phosphodiesterase	3.6	1.8
AF023621_at	sortilin	3.5	2
rc_AI639246_at		3.2	1.7
Rc_AA957003_at		3.2	1.6
L00603_at	vesicular monoamine transporter	3	2.4
U13396_at	protein-tyrosine kinase (JAK2)	3	2.1
M64986_g_at	amphoterin mRNA	3	1.5
L11319_at	five-lipoxygenase activating protein (FLAP)	2.8	1.5
rc_AA892851_g_at		2.7	2.3
X78605_at	rab4b mRNA for ras-homologous GTPase	2.7	2.3
U49930_g_at	ICE-like cysteine protease (Lice)	2.7	1.6
rc_AA893534_at		2.6	1.8
D17521_at	protein kinase C-regulated chloride channel	2.6	1.7
U27201_at	tissue inhibitor of metalloproteinase 3 (TIMP-3)	2.6	1.6
M55532_at	carbohydrate binding receptor	2.5	1.8
D13962_g_at	neuron glucose transporter (GLUT3)	2.5	1.4
rc_AA893664		2.3	1.8
AJ000557cds_s_at	Janus protein tyrosine kinase 2, JAK2	2.2	1.6
rc_AA875206_at		2.2	1.5
D84346_s_at	Nap1 protein	2.2	1.4
rc_AA800275_at		2.2	1.4
rc_AI171962_s_at		2.2	1.4
S70011_g_at	tricarboxylate carrier	2.1	1.8
AF084186_s_at	alpha-fodrin (A2A)	2.1	1.7
L25387_g_at	phosphofructokinase C (PFK-C)	2.1	1.6
rc_AA892049_at		2.1	1.4
rc_AI638939_at		2.1	1.4
U09631_at	VIP2 vasoactive intestinal peptide receptor	2.1	1.4
M93017_at	Rat alternatively spliced mRNA	2.1	1.3
rc_AA799402_at		2	1.8
X78949_at	prolyl 4-hydroxylase alpha subunit	2	1.7
rc_AA799650_at		2	1.6
rc_AA859520_at		2	1.6
U41164_at	Cys2/His2 zinc finger protein (rKr1)	2	1.6
X63995_at	NTT	2	1.6
L01793_at	glycogenin	2	1.3
rc_AA891732_at		1.9	1.5
rc_AA892511_at		1.9	1.5
rc_AI230778_at		1.9	1.5
AF099093_g_at	ubiquitin-conjugating enzyme UBC7	1.9	1.4
rc_AA893217_at		1.9	1.4
rc_AA956958_at		1.9	1.4
rc_AI045794_at		1.9	1.3
rc_AA799637_at		1.8	1.6
rc_H31610_at		1.8	1.5
X78606_at	rab28 mRNA for ras-homologous GTPase	1.8	1.5
rc_AA875594_s_at		1.8	1.4
rc_AI171506_g_at		1.8	1.4
S70011_at	tricarboxylate carrier	1.8	1.4
rc_AA893002_at		1.8	1.3
X61295cds_s_at	L1 retroposon, ORF2 mRNA	1.8	1.3
rc_AA799570_at		1.7	1.5
rc_AA874934_at		1.7	1.5
rc_AA892642_at		1.7	1.4
X63253cds_s_at	serotonin transporter	1.7	1.4
rc_AA800787_at		1.7	1.3
rc_AA891068_f_at		1.7	1.3
rc_AA892014_r_at		1.7	1.3
rc_AA892496_at		1.7	1.3
rc_AA893237_at		1.7	1.3
rc_AI228247_at		1.7	1.3
rc_AI639162_at		1.6	1.5
X73371_at	Fc gamma receptor	1.6	1.4
rc_AA801286_at		1.4	1.3
U57050_g_at	hypertension-related mRNA	1.3	1.3

Table 4 sets forth genes induced in the blood of rats 24 hours following the sham control operation (n=3 rats) as compared with genes expressed in the blood of untouched control rats (n=3 rats). The accession number of the gene is given, the name of the gene is given where known, the average fold induction is given, as well as the minimum fold induction is given for each gene. A number of the genes are ESTs that have not yet been subjected to a BLAST search. This list was produced from arrays that contained over 8000 genes.

TABLE 4


Accession No.	Name	Average	Minimum


M58040_at	transferrin receptor	5.8	3
D50564_at	mercaptopyruvate sulfurtransferase	5	1.55
U07201_at	asparagine synthetase	4	3
rc_AA894273_at		3	1.7
AF087674_at	insulin receptor substrate 2 (IRS-2)	2.9	1.9
rc_AA858607_at		2.7	1.3
X06827_at	porphobilinogen deaminase	2.6	1.6
D28966_at	prostacyclin receptor	2.6	1.5
rc_AA852046_s_at		2.6	1.3
E00594cds_at	immunoglobulin E binding factor activity	2.5	1.4
	peptide
M91235_f_at	VL30 element	2.4	1.8
rc_AA892897_at		2.3	1.5
M91234_f_at	VL30 element	2.2	1.5
rc_AA819793_at		2.1	1.7
U12514_at	transcriptional regulator MSX-2 (MSX-2)	2.1	1.4
AF079162_at	patched (ptc)	2.1	1.3
X67948_at	channel integral membrane protein 28	2.1	1.3
X82396_at	cathepsin B	2	1.6
AB015645_at	G protein-coupled receptor	1.9	1.5
L12384_at	ADP-ribosylation factor 5	1.9	1.3
AF087696_at	dlg 2	1.8	1.4
U53486mRNA_s_at	corticotropin releasing factor receptor	1.8	1.4
rc_AA800566_g_at		1.8	1.3
X12554cds_s_at	heart cytochrome c oxidase subunit VIa	1.8	1.3
X63722cds_s_at	vascular cell adhesion molecule-1	1.4	1.2

The above blood data only catalogues the genes that show an increase of expression in one condition versus the other. Not listed above are an equal number of genes that show down-regulation or decreases following stroke, seizures and hypoxia when compared to controls. The genes that show down regulation are just as important for describing the pattern of gene regulation in blood but are not included the downregulated genes in the above lists for the sake of simplicity. The downregulated genes in the list of hypoxia-regulated genes in brain are set forth below as an example.

The above data show that different genes, for the most part, are induced in the blood cells of rats following stroke, hypoxia and status epilepticus as compared with the controls. In addition, the genes induced in the blood cells of rats following sham control operations differed from the genes expressed in the blood cells of untouched rats. This data suggests that different patterns of expression will occur in the blood depending on the injury or the cause of the injury. The pattern of expression for each injury is distinct and therefore can be used to assess the injury.

In further support, the following Tables 5 and 6 list those genes induced in the brain following stroke, kainic induced seizures, and hypoxia as compared with untouched controls and sham-operated controls. This data supports the concept that gene expression in the brain differs following different types of injury, just as gene expression in the blood differs following different types of injury.

TABLE 5


			Kainic Acid
		Stroke Ischemia	Seizure	Hypoxia
Probe Set	Name	(fold change)	(fold change)	(fold change)


M86389cds_s _—	Rat hsp 27	361.9	309.2	NC
S82649-r-at	Narp + neuronal activity-regulated	251.8	72.5	NC
	pentraxin
rc_AI169327_g _—	Tissue Inhibitor of	239	186.7	NC
	Metalloproteinase
z27118cds_s _—	Rat hsp 70	183.4	37.3	NC
aa848563_s_a	heat shock protein 70	145	27.1	NC
d00753_at	Rat RNA for contrapsin-like	134.4	55.4	NC
	protoease inhibitor related protein
	(CPi-26)
m14656_at	osteopontin m RNA	79.3	39	NC
x17053RNA_s	rat immediate-early serum response	67.2	51.3	NC
	gene
jo2722cds_at _—	Rat heme oxygenase gene	68.5	20.2	NC
z75029_s_at	R. norvegicus hsp 70.2 RNA for heat	64.6	12.3	NC
	shock protein 70
m36317_s_at	Rat thyrotropin-releasing hormone	63.5	30.4	NC
	(TRH) precursor
rc_aa998683	heat shock protein 27	60.6	50.5	NC
ab002588_at	glycerol 3-phosphate deyydrogenase	53.3	52.4
m23566exon_s _—	alpha-2-macroglobulin gene	53.2	NC	NC
rc_ai045030	C/EBP	52	21	NC
x07266_cds_s _—	Rat RNA for gene 33 polypeptide	51.7	21.7	NC
af028784RNA	GFAP	49.7	52.2	NC
af025308_f_a	Rattus norvegicus MHC class 1b	44.4	no	NC
	antigen (RT1.Cl) gene
m61875_s_at	CD44	41.8	69.4	NC
x76454_at	ri1 RNA	39.8	50.3	NC
rc_aa818604		37.4	7.2	NC
s71196RNA_s _—	BDNF	35.8	NC	NC
M23643cds_s _—	TRH	35.1	12	NC
x59864RNA_a	Rat ASM15 gene	34	52.2	NC
m26744_at	interleukin 6 (IL6) RNA	32.2	NC	NC
L16764_s_at	heat shock rotein 70 (HSP70)	32.2	10.5	NC