Patent Application
20100267582
This application relates to methods of evaluating an ischemic stroke, methods of identifying a treatment modality for a subject who has had an ischemic stroke, methods of identifying compounds that alter the activity of an ischemic stroke-related molecule, as well as arrays and kits that can be used to practice the disclosed methods.
Stroke is the third leading cause of death and the leading cause of adult disability in developed countries (Simons et al., Stroke 29:1341-6, 1998; Adams et al., Ischemic Cerebrovascular Disease. New York: Oxford, 2001). Strokes are caused by an interruption of blood flow to the brain, by either an intravascular occlusion (such as an arterial thrombus) or a hemorrhage. The American Heart Association estimates that there are approximately three million stroke survivors in the United States, most of whom are disabled. Despite the prevalence and burden of this disease, stroke precipitants and pathophysiological mechanisms in individual patients are often unknown. It is also difficult to accurately predict whether a stroke will lead to only minor neurological sequelae or more serious medical consequences.
Gene expression profiling involves the study of mRNA levels in a tissue sample to determine the expression levels of genes that are expressed or transcribed from genomic DNA. Animal experiments in focally ischemic brain tissue have indicated that there are alterations in gene expression following a stroke (Stenzel-Poore et al., Lancet 362:1028-37, 2003; Lu et al., J. Cereb. Blood Flow. Metab. 23:786-810, 2003; Tang et al., Eur J Neurosci 15:1937-52, 2002; Tang et al., Ann. Neurol. 50:699-707, 2001; and Tang et al., J Cereb Blood Flow Metab 23:310-9, 2003). However, gene expression profiling has not yet been applied to clinical human stroke, primarily because brain tissue samples are inaccessible and rarely justified. Therefore, an assay that can allow one to determine the genetic expression profile of ischemic stroke without the need for brain tissue samples is needed.
Currently, there is no specific blood marker of acute stroke. Following a stroke, released brain antigens can be detected in the blood. Such antigens include S100B, neuron specific enolase (NSE), and glial fibrillary acid protein (GFAP), although S100B and GFAP are of low sensitivity for early stroke diagnosis, and NSE and myelin basic protein (MBP) MBP are non-specific (Lamers et al., Brain. Res. Bull. 61:261-4, 2003). Four soluble factors that have demonstrated moderate sensitivity and specificity for the diagnosis of stroke include two markers of inflammation (matrix metalloproteinase-9 and vascular cell adhesion molecule), one marker of glial activation (S100beta) and one thrombosis marker (von Willebrand factor) (Lynch et al., Stroke 35:57-63, 2004). However, a panel of markers which allow one to diagnose and prognose ischemic stroke with high diagnostic sensitivity and specificity is still needed.
Although stroke is one of the leading causes of morbidity and mortality in developed countries, methods for rapidly and accurately determining whether a subject has had a stroke are expensive and invasive. Therefore, new methods are needed for evaluating a stroke, for example for determining whether an ischemic stroke has occurred, for determining the severity of the stroke or the likely neurological recovery of the subject who had an ischemic stroke, or combinations thereof. In particular examples, the disclosed methods offer a potentially lower cost alternative to expensive imaging modalities (such as MRI and CT scans), can be used in instances where those imaging modalities are not available (such as in field hospitals), and can be more convenient than placing individuals in scanners (for example for subjects who can not be subjected to MRI, such as those having certain types of metallic implants in their bodies).
Using these methods, appropriate therapy protocols for subjects who have had an ischemic stroke can be identified and administered. For example, because the results of the disclosed methods are highly reliable predictors of the ischemic nature of the stroke, the results can also be used (alone or in combination with other clinical evidence and brain scans) to determine whether thrombolytic therapy designed to lyse a neurovascular occlusion such as a thrombus (for example by using tissue plasminogen activator or streptokinase) should be administered to the subject. In certain examples, thrombolytic therapy is given to the subject once the results of the differential expression assay are known if the assay provides an indication that the stroke is ischemic in nature.
The inventors have identified changes in gene expression in peripheral blood mononuclear cells (PBMCs) that allow one to evaluate a stroke, for example to determine whether a subject has had an ischemic stroke, to determine the severity of an ischemic stroke, to determine the likely neurological recovery of the subject, or combinations thereof. The disclosed methods allow one to screen many genes simultaneously and serially and only a relatively small amount of cell or tissue sample is needed. Changes in gene expression were observed in at least 22 genes, at least 82 genes, at least 190 genes, or even at least 637 genes depending on sensitivity and specificity used. In particular examples, subjects who had an ischemic stroke showed increased gene expression in CD163; hypothetical protein FLJ22662 Laminin A motif; bone marrow stromal cell antigen 1 (BST-1, also known as CD157); Fc fragment of IgG, high affinity Ia, receptor for (FcγRI, also known as CD64); baculoviral IAP repeat-containing protein 1 (also referred to in the literature as neuronal apoptosis inhibitory protein); or KIAA0146, or any combinations thereof, such as a change in expression in at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 of these genes. In some examples, subjects who had an ischemic stroke showed increased gene expression in four classes of genes: genes involved in white blood cell activation and differentiation, genes related to hypoxia, genes involved in vascular repair, and genes related to a PBMC response to the altered cerebral microenvironment.
The disclosed gene expression fingerprint of ischemic stroke enables methods of evaluating a stroke, for example determining whether a subject had an ischemic stroke, determining the prognosis of a subject who had an ischemic stroke, as well as determining an appropriate treatment regimen for a subject who had an ischemic stroke. In some examples, the disclosed methods are at least 78% sensitive and at least 80% specific for identifying those subjects who have suffered an ischemic stroke, for example within the past 72 hours. In other examples, the disclosed methods are at least 80% sensitive (such as at least 85% sensitive or at least 90% sensitive) and at least 80% specific (such as at least 85% specific or at least 90% specific) for identifying those subjects who have suffered an ischemic stroke, for example within the past 72 hours. In particular examples, the disclosed methods are at least 80% sensitive for predicting the likelihood of neurological recovery of the subject.
In some examples, the method involves detecting patterns of increased protein expression, decreased protein expression, or both. Such patterns of expression can be detected either at the nucleic acid level (such as quantitation of mRNAs associated with protein expression) or the protein level (such as quantitative spectroscopic detection of proteins). Certain methods involve not only detection of patterns of expression, but detection of the magnitude of expression (increased, decreased, or both), wherein such patterns are associated with the subject having had an ischemic stroke, or is associated with predicted clinical sequelae, such as neurological recovery following an ischemic stroke.
The disclosed methods are the first that permit accurate diagnosis of an ischemic stroke using PBMCs with high sensitivity and specificity. PBMCs infiltrate the evolving cerebral infarct as part of the tissue remodeling process. Release of brain antigens from damaged neural cells may allow sensitization of PBMCs followed by changes in functional gene expression.
The disclosed methods can be performed on a subject who is suspected of having had a stroke, for example prior to radiographic investigation. In another example, the method is performed on a subject known to have had a stroke, as the disclosed assays permit early and accurate stratification of risk of long-lasting neurological impairment.
In one example, the method of evaluating a stroke includes determining whether a subject has changes in expression in four or more ischemic stroke-associated molecules that comprise, consist essentially of, or consist of, sequences (such as a DNA, RNA or protein sequence) involved in white blood cell activation and differentiation, sequences related to hypoxia, sequences involved in vascular repair, and sequences related to a PBMC response to the altered cerebral microenvironment, such as those listed in Table 5.
In other examples, ischemic stroke-associated molecules comprise, consist essentially of, or consist of, CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, or any 1, 2, 3, 4, 5, or 6 of these molecules. For example, ischemic stroke-associated molecules can comprise, consist essentially of, or consist of, 4 or more, such as 5 or more, 10 or more, 20 or more, 22 or more, 50 or more, 75 or more, 80 or more, 82 or more, 100 or more, 150 or more, 190 or more, 200 or more, 300 or more, 500 or more, 600 or more, or 637 or more of the nucleic acid or protein sequences listed in Tables 2-5. Any of the identified sequences can be used in combination with such sets or subsets of sequences.
In a particular example, evaluating a stroke includes detecting differential expression in at least four ischemic stroke-related molecules of the subject, such as any combination of at least four genes (or the corresponding proteins) listed in any of Tables 2-5, wherein the presence of differential expression of at least four ischemic-stroke related molecules indicates that the subject has had an ischemic stroke. Therefore, such methods can be used to diagnose an ischemic stroke. In particular examples, the at least four ischemic-stroke related molecules include at least one of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, such as at least 2, at least 3, at least 4, at least 5 or at least 6 of such molecules. For example, the method can include determining if the subject has increased gene (or protein) expression of at least one of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, optionally in combination with determining if the subject has altered gene (or protein) expression of any other combination of other ischemic stroke-associated molecules, such as any combination of at least 3 other genes (for example any combination of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, or even at least 500 genes) listed in Tables 2-5.
In a particular example, differential expression is detected by determining if the subject has increased gene (or protein) expression of at least one of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, and determining if the subject has decreased gene (or protein) expression of at least one of intercellular adhesion molecule 2, protein kinase D2, GATA binding protein 3, hypothetical protein FLJ20257, or protein kinase C, theta. For example, differential expression can be detected by determining if the subject has increased gene (or protein) expression of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, and determining if the subject has decreased gene (or protein) expression of intercellular adhesion molecule 2, protein kinase D2, GATA binding protein 3, hypothetical protein FLJ20257, and protein kinase C, theta.
In one example, the method includes determining if the subject has an increase in gene expression in any combination of at least four of the genes listed in Table 5, for example an increase in at least 5, at least 10, at least 15, at least 20, or at least 22 of the genes listed in Table 5. An increase in expression in any combination of four or more of the genes listed in Table 5 (or the corresponding proteins), and particularly any combination of at least one gene (or protein) from each of the four classes of genes listed in Table 5 (such as any combination of at least 2 or at least 3 sequences from each of the four classes of genes listed in Table 5) indicates that the subject has had an ischemic stroke. Any one of the set of genes (or proteins) can be identified by a single one or the genes (or proteins) listed in Table 5. Any one of the genes (or proteins) in Table 5 can be combined with any other combination of the genes (or proteins) in Table 5 to produce a combination or subcombination of genes (or proteins).
In one example, the method of evaluating a stroke includes determining if the subject has a change in gene expression (such as an increase or decrease) in any combination of at least 150 of the 190 of the genes listed in Table 3, for example a change in expression in at least 160, at least 170, at least 175, at least 180, or at least 185 of the genes listed in Table 3. Any one of the set of genes can be identified by a single one or the genes listed in Table 3. Any one of the genes (or proteins) in Table 3 can be combined with any other combination of the genes (or proteins) in Table 3 to produce a combination or subcombination of genes. A change in expression in any combination of 150 or more of the genes listed in Table 3 (or the corresponding proteins) indicates that the subject has had an ischemic stroke.
In another example, the method of evaluating a stroke includes determining if the subject has a change in gene expression (such as an increase or decrease) in any combination of at least 510 of the 637 of the genes listed in Table 2, for example an increase or decrease in any combination of at least 510, at least 550, at least 575, at least 600, at least 620, or at least 630 of the genes listed in Table 2. Any one of the set of genes (or proteins) can be identified by a single one or the genes (or proteins) listed in Table 2. Any one of the genes (or proteins) in Table 2 can be combined with any other combination of the genes (or proteins) in Table 2 to produce a combination or subcombination of genes. A change in expression in any combination of 510 or more of the genes listed in Table 2 (or the corresponding proteins) indicates that the subject has had an ischemic stroke.
In some examples, the amount of gene (or protein) expression in the subject is compared to a control, such as the gene (or protein) expression of a subject who has not had an ischemic stroke, wherein an increase or decrease in expression in any combination of four or more ischemic stroke related genes listed in Tables 2-5 compared to the control indicates that the subject has experienced an ischemic stroke. For example, an increase in expression in any combination of four or more ischemic stroke related genes (or the corresponding proteins) listed in Table 5, such as at least one gene (or the corresponding protein) from each class listed in Table 5, compared to the control indicates that the subject has experienced an ischemic stroke.
In particular examples evaluating the stroke includes predicting a likelihood of severity of neurological sequelae of the ischemic stroke. In some examples, evaluating the stroke includes predicting a likelihood of neurological recovery of the subject. For example, if there is differential expression (such as increased expression) in at least the 22 ischemic-stroke related molecules listed in Table 5, indicates that the subject has a higher risk of long-term adverse neurological sequelae and therefore a lower likelihood of neurological recovery. In another example, detecting a change in expression in any combination of 150 or more of the genes listed in Table 2 or 3 (or the corresponding proteins) indicates that the subject has a higher risk of long-term adverse neurological sequelae and therefore a lower likelihood of neurological recovery. In yet another example, detecting a change in expression in any combination of at least 500 of the 637 of the genes listed in Table 2, for example an increase or decrease in any combination of at least 510, at least 550, at least 575, at least 600, at least 620, or at least 630 of the genes listed in Table 2 indicates that the subject has a higher risk of long-term adverse neurological sequelae and therefore a lower likelihood of neurological recovery. In some examples, differential expression in the subject is compared to differential expression of a subject who has not had an ischemic stroke, wherein a change in expression in at least the 22 ischemic-stroke related molecules listed in Table 5, such as any combination of 150 or more of the genes listed in Tables 2 or 3 (or the corresponding proteins) compared to the control indicates that the subject has a higher risk of long-term adverse neurological sequelae and therefore a lower likelihood of neurological recovery.
The disclosed methods can further include administering to a subject a treatment to avoid or reduce ischemic injury if the presence of differential expression indicates that the subject has had an ischemic stroke. For example, a change in expression in at least four ischemic stroke related molecules, such as a combination that includes at least four of the molecules listed in Tables 2-5, indicates that the subject has had an ischemic stroke (and not a hemorrhagic stroke) and is in need of thrombolytic therapy (such as t-PA or heparin), anticoagulant therapy (such as coumadin), or combinations thereof. Therefore, the disclosed methods differentiate ischemic from hemorrhagic stroke, and allow one to administer the appropriate therapy to the subject. In some examples, the amount of differential expression in the subject is compared to the expression of a subject who has not had an ischemic stroke, wherein a change in expression in at least four ischemic stroke related molecules listed in Table 2-5 (or the corresponding proteins), such as at least those 22 listed in Table 5, compared to the control indicates that the subject would benefit from thrombolytic therapy, anticoagulant therapy, or combinations thereof.
In some examples the presence of differential expression is evaluated by determining a t-statistic value that indicates whether a gene or protein is up- or down-regulated. For example, an absolute t-statistic value can be determined. In some examples, a negative t-statistic indicates that the gene or protein is downregulated, while a positive t-statistic indicates that the gene or protein is upregulated. In particular examples, a t-statistic less than −3 indicates that the gene or protein is downregulated, such as less than −3.5, less than −3.6, less than −3.7 or even less than −3.8, while a t-statistic of at least 3, such as at least 3.5, at least 3.7, or at least 3.8 indicates that the gene or protein is upregulated.
Differential expression can be detected at any time following the onset of clinical signs and symptoms that indicate a potential stroke, such as within 24 hours, within 7-14 days, or within 90 days of onset of clinical signs and symptoms that indicate a potential stroke. Examples of such signs and symptoms include, but are not limited to: headache, sensory loss (such as numbness, particularly confined to one side of the body or face), paralysis (such as hemiparesis), pupillary changes, blindness (including bilateral blindness), ataxia, memory impairment, dysarthria, somnolence, and other effects on the central nervous system recognized by those of skill in the art.
In particular examples, the disclosed methods include isolating nucleic acid molecules from PBMCs of a subject suspected of having had an ischemic stroke (or known to have had an ischemic stroke), such as mRNA molecules. The isolated nucleic acid molecules are contacted with or applied to an array, for example an array that includes oligonucleotide probes capable of hybridizing to ischemic stroke-associated genes. In another particular example, the disclosed methods include purifying proteins from PBMCs of a subject suspected of having had an ischemic stroke (or known to have had an ischemic stroke). The isolated proteins are contacted with or applied to an array, for example an array that includes antibody probes capable of hybridizing to ischemic stroke-associated proteins. In some examples, PBMCs are obtained within at least the previous 72 hours of a time when the stroke is suspected of occurring, such as within the previous 24 hours.
Also provided herein are arrays that include molecules that permit evaluation of a stroke. Such arrays in particular examples permit quantitation of ischemic stroke-related nucleic acid or protein sequences present in a sample, such as a sample that includes PBMC nucleic acid molecules or proteins.
In one example, the array includes oligonucleotide probes capable of hybridizing to nucleic acid molecules (such as gene, cDNA or mRNA sequences) involved in white blood cell activation and differentiation, nucleic acid molecules related to hypoxia, nucleic acid molecules involved in vascular repair, and nucleic acid molecules related to a PBMC response to the altered cerebral microenvironment, such as at least those listed in Table 5. Examples of particular genes are provided in Tables 2-5. In particular examples, the array includes probes that recognize any combination of at least 4 of the genes listed in any of Tables 2-5, for example at least 10, at least 20, at least 50, at least 100, at least 150, at least 160, at least 170, at least 175, at least 180, at least 185, at least 200, at least 400, at least 500, at least 510, at least 550, at least 575, at least 600, at least 620, or at least 630 of the genes listed in any of Tables 2-5. For example, the array can include oligonucleotide probes capable of hybridizing to a sequence that encodes at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, or any one of these. In one example, the array includes oligonucleotide probes capable of hybridizing to a sequence that encodes one or more of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, such as at least 2, at least 3, at least 4, at least 5 or at least 6 of such molecules. In some examples, the array includes probes that recognize any combination of at least one gene from each of the four classes listed in Table 5, such as at least 2, at least 3, at least 5, or at least 10 genes from each class.
The isolated nucleic acid molecules are incubated with the array for a time sufficient to allow hybridization between the isolated nucleic acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecules:oligonucleotide probe complexes. The isolated nucleic acid molecules:oligonucleotide probe complexes are then analyzed to determine if there are changes in gene expression (such as increases or decreases), for example changes in expression of any combination of four or more of the genes listed in Table 5, such as 20 or more of the genes listed in Tables 2-5, or such as 150 or more of the genes listed in Tables 2-4. In particular examples, changes in gene expression are quantitated. The presence of increased expression of four or more genes listed in Tables 2-5 with a positive t-statistic value, or decreased expression of four or more genes listed in Tables 2-4 with a negative t-statistic value (or any combination thereof, such as decreased expression of at least one gene and increased expression of at least 3 genes listed in Tables 2-4), after multiple comparison correction, indicates that the subject has had an ischemic stroke.
In another example, the method includes isolating proteins from PBMCs of a subject suspected of having had an ischemic stroke, or known to have had an ischemic stroke. In particular examples the assay is performed on substantially purified or isolated PBMCs that have been separated, for example, for other leukocytes in the blood. The isolated proteins are contacted with or applied to an array.
Arrays that can be used to detect and quantitate proteins for evaluating stroke are also provided. For examples, the array, such as a protein-binding array, can include probes (such as an oligonucleotide probes or antibodies) capable of hybridizing to ischemic-stroke related proteins, such as proteins involved in white blood cell activation and differentiation, proteins related to hypoxia, proteins involved in vascular repair, and proteins related to a PBMC response to the altered cerebral microenvironment. Examples of particular ischemic-stroke related proteins are provided in Tables 2-5. The isolated proteins are incubated with the array for a time sufficient to allow hybridization between the proteins and probes on the array, thereby forming protein:probe complexes.
The protein:probe complexes are then analyzed and in some examples quantitated to determine if there are changes in gene expression (such as increases or decreases) in any combination of four or more of the molecules listed in any of Tables 2-5, such as changes in expression of one or more of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-vcontaining protein 1; and KIAA0146, or 2, 3, 4, or 5 of these. In a specific example, protein:probe complexes are analyzed (for example quantitated) to determine if there are increases in expression in any combination of at least one protein from each of the four classes listed in Table 5, such as at least 2, at least 3, at least 5, or at least 10 genes from each of the classes listed in Table 5. The presence of increased or decreased expression of any combination of four or more proteins listed in Tables 2-4 (or increased expression of any combination of four or more proteins listed in Table 5), indicates that the subject has had an ischemic stroke.
In particular examples, the disclosed arrays are capable of evaluating a stroke, for example for determining whether a subject has had an ischemic stroke, determining the severity of the ischemic stroke, determining the likelihood of neurological recovery of a subject who had an ischemic stroke, determining how to treat a subject who had an ischemic stroke, or combinations thereof. Such arrays include oligonucleotides that are complementary to ischemic stroke-related genes, such as those involved in white blood cell activation and differentiation, genes related to hypoxia, genes involved in vascular repair, and genes related to a PBMC response to the altered cerebral microenvironment. Examples of particular genes are provided in Tables 2-5. Kits including such arrays are also disclosed.
In one example, proteins a biological sample are quantitated, for instance by quantitative mass spectroscopy, to determine whether proteins associated with ischemic stroke or prognosis of ischemic stroke are upregulated, downregulated, or both.
Also provided in the present disclosure are methods of identifying one or more agents that alter the activity (such as the expression) of an ischemic stroke-related molecule (for example a gene or protein), such as one or more of those listed in Tables 2-5. If desired, multiple test agents and multiple ischemic stroke-related molecules can be screened at the same time. In one example, the method is used to screen the effect of one test agent on multiple ischemic stroke-related molecules simultaneously (such as all of the ischemic stroke-related molecules listed in Table 2 or Table 3). In another example, the method is used to screen the effect of multiple test agents on one ischemic stroke-related molecule, such as one of the molecules listed in Tables 2-5. In particular examples, the identified agent alters the activity of an ischemic stroke-related molecule that is upregulated or downregulated following an ischemic stroke. For example, the agent can normalize activity of an ischemic stroke-related molecule that is upregulated or downregulated following an ischemic stroke, such as by increasing the activity of an ischemic stroke-related molecule that is downregulated following an ischemic stroke, or decreasing activity of an ischemic stroke-related molecule that is upregulated following an ischemic stroke. The disclosed methods can be performed in vitro (for example in a cell culture) or in vivo (such as in a mammal).
In one example, the test agent is an agent in pre-clinical or clinical trials or approved by a regulatory agency (such as the Food and Drug Administration, FDA), to treat ischemic stroke. For example, the method can be used to determine if the agent alters the activity of one or more ischemic stroke-related molecules that modifies response to treatment and can predict the best responders.
In another example, the method is used to identify a particular class of agents, such as those that are effective against hypoxia. For example, one or more test agents can be screened using the methods disclosed herein, and differential expression of the disclosed hypoxia-related genes (or proteins) measured. Test agents that alter the activity of one or more disclosed hypoxia-related molecules are candidates for treatment of hypoxia.
The disclosed methods can also be used in toxicogenomics, for example to identify genes or proteins whose expression is altered in response to medication-induced toxicity and side-effects. In one example, the disclosed ischemic stroke-related molecules are screened to identify those whose activity is altered in response to an agent. For example, the disclosed ischemic stroke-related molecules can be used determine if an agent promotes or induces ischemic stroke. Briefly, the test agent is contacted with a normal cell (such as a PBMC, endothelia, or neuronal cell), such as a cell that has not been exposed to conditions that mimic an ischemic stroke, and differential expression of one or more ischemic stroke molecules measured using the methods disclosed herein. If the agent promotes or induces differential expression of one or more, such as at least 4 of the disclosed ischemic stroke-related molecules (such as those listed in Tables 2-5) in an otherwise normal cell or mammal (for example as compared to a similar cell cultured in similar conditions without the test agent), this indicates that the agent may cause or promote an ischemic stroke in vivo. Such a result may indicate that further studies of the agent are needed. In another example, cells from a subject who is to receive a pharmaceutical agent are obtained (such as PBMCs), and the pharmaceutical agent incubated with the cells as described above, to determine if the pharmaceutical agent causes or promotes differential expression of one or more ischemic stroke-related molecules. Such a result would indicate that the subject may react adversely to the agent, or that a lower dose of the agent should be administered.
The disclosure also provides methods of generating a brain imaging tracer or white blood cell tracers for molecular imaging, such as imaging to determine if a subject has had an ischemic stroke. Briefly, a labeled antibody that recognizes an ischemic stroke-related molecule, such as those involved in white blood cell activation and differentiation, those involved in the response to altered cerebral microenvironment, or combinations thereof (see Table 5). In one example, the label is a fluorophore, radioisotope, or other compound that can be used in diagnostic imaging, such as a nuclear medicine radio-isotope (for example 99mTechnetium for use with single photon emission computed tomography, 18Fluorodeoxyglucose (18FDG) for use with positron emission tomography, or a paramagnetic contrast agent for magnetic resonance imaging). The labeled antibody can be administered to the subject, for example intravenously, and the subject imaged using standard methods.
The foregoing and other features and advantages of the disclosure will become more apparent from the following detailed description of a several embodiments.
The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID NOS: 1-2 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of adrenomedullin.
SEQ ID NOS: 3-4 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of CD14.
SEQ ID NOS: 5-6 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of CD36.
SEQ ID NOS: 7-8 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of caspase 1.
SEQ ID NOS: 9-10 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of a-Catenin.
SEQ ID NOS: 11-12 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of FcR2a.
SEQ ID NOS: 13-14 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of FcER1a.
SEQ ID NOS: 15-16 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of cathepsin B.
SEQ ID NOS: 17-18 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of TRL2.
SEQ ID NOS: 19-20 are oligonucleotide sequences used to perform RT-PCR to determine expression levels of INFGR1.
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. For example, the term “comprising a nucleic acid molecule” includes single or plural nucleic acid molecules and is considered equivalent to the phrase “comprising at least one nucleic acid molecule.” The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
PBMC: peripheral blood mononuclear cell
Real time-PCR: real time polymerase chain reaction
Administration: To provide or give a subject an agent by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
Amplifying a nucleic acid molecule: To increase the number of copies of a nucleic acid molecule, such as a gene or fragment of a gene, for example a region of a ischemic stroke-associated gene. The resulting products are called amplification products.
An example of in vitro amplification is the polymerase chain reaction (PCR), in which a biological sample obtained from a subject (such as a sample containing PBMCs) is contacted with a pair of oligonucleotide primers, under conditions that allow for hybridization of the primers to a nucleic acid molecule in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule. Other examples of in vitro amplification techniques include quantitative real-time PCR, strand displacement amplification (see U.S. Pat. No. 5,744,311); transcription-free isothermal amplification (see U.S. Pat. No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Pat. No. 5,427,930); coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); and NASBATM RNA transcription-free amplification (see U.S. Pat. No. 6,025,134).
Quantitative real-time PCR is another form of in vitro amplifying nucleic acid molecules, enabled by Applied Biosystems (TaqMan PCR). Real-time quantitative TaqMan PCR has reduced the variability traditionally associated with quantitative PCR, thus allowing the routine and reliable quantification of PCR products to produce sensitive, accurate, and reproducible measurements of levels of gene expression. The 5′ nuclease assay provides a real-time method for detecting only specific amplification products. During amplification, annealing of the probe to its target sequence generates a substrate that is cleaved by the 5′ nuclease activity of Taq DNA polymerase when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerization ensures that cleavage of the probe occurs only if the target sequence is being amplified. The use of fluorogenic probes makes it possible to eliminate post-PCR processing for the analysis of probe degradation. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by Förster resonance energy transfer (FRET) through space. Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5′ end and the quencher at the 3′ end.
Anti-coagulants: Agents that decrease or prevent blood clotting. Anticoagulants can avoid the formation of new clots, and prevent existing clots from growing (extending), for example by decreasing or stopping the production of proteins necessary for blood to clot. Examples include, but are not limited to, aspirin, heparin, ximelagatran, and warfarin (Coumadin). Administration of anticoagulants is one treatment for ischemic stroke, for example to prevent further strokes. A particular type of anti-coagulant are anti-platelet agents, which can also be used to prevent further strokes from occurring and include aspirin, clopidogrel (Plavix), aspirin/dipyridamole combination (Aggrenox), and ticlopidine (Ticlid). Other agents used to prevent stroke recurrence are antihypertensive drugs and lipid-lowering agents such as statins.
Array: An arrangement of molecules, such as biological macromolecules (such as peptides or nucleic acid molecules) or biological samples (such as tissue sections), in addressable locations on or in a substrate. A “microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. Arrays are sometimes called DNA chips or biochips.
The array of molecules (“features”) makes it possible to carry out a very large number of analyses on a sample at one time. In certain example arrays, one or more molecules (such as an oligonucleotide probe) will occur on the array a plurality of times (such as twice), for instance to provide internal controls. The number of addressable locations on the array can vary, for example from at least four, to at least 10, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, least 550, at least 600, at least 800, at least 1000, at least 10,000, or more. In particular examples, an array includes nucleic acid molecules, such as oligonucleotide sequences that are at least 15 nucleotides in length, such as about 15-40 nucleotides in length. In particular examples, an array includes oligonucleotide probes or primers which can be used to detect ischemia stroke-associated sequences, such as any combination of at least four of those listed in Table 5, such as at least 10, at least 20, at least 50, at least 100, at least 150, at least 160, at least 170, at least 175, at least 180, at least 185, at least 200, at least 400, at least 500, at least 510, at least 550, at least 575, at least 600, at least 620, or at least 630 of the sequences listed in any of Tables 2-5. In some examples, an array includes oligonucleotide probes or primers which can be used to detect at least one gene from each of the four classes of genes listed in Table 5, such as at least 2, at least 3, at least 5, or even at least 10 genes from each of the four classes of genes listed in Table 5.
Within an array, each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array. The feature application location on an array can assume different shapes. For example, the array can be regular (such as arranged in uniform rows and columns) or irregular. Thus, in ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position. Often, ordered arrays are arranged in a symmetrical grid pattern, but samples could be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters). Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.
Protein-based arrays include probe molecules that are or include proteins, or where the target molecules are or include proteins, and arrays including nucleic acids to which proteins are bound, or vice versa. In some examples, an array contains antibodies to ischemic stroke-associated proteins, such as any combination of at least four of those listed in Table 5, such as at least 10, at least 20, at least 50, at least 100, at least 150, at least 160, at least 170, at least 175, at least 180, at least 185, at least 200, at least 400, at least 500, at least 510, at least 550, at least 575, at least 600, at least 620, or at least 630 of the sequences listed in any of Tables 2-5. In particular examples, an array includes antibodies or proteins that can detect at least one protein from each class listed in Table 5, such as at least 2, at least 3, at least 5, or even at least 10 genes from each class listed in Table 5.
Baculoviral IAP repeat-containing protein 1 (Birc1): A protein that includes one or more baculoviral IAP repeat (BIR) domains, which is capable of decreasing (an in some examples inhibiting) the biological activity of caspases, and in some examples thereby decreasing or inhibiting apoptosis. The term baculoviral IAP repeat-containing protein 1 includes any Birc1 gene, cDNA, mRNA, or protein from any organism and that is a Birc1 that can decrease or inhibit caspase biological activity. Also referred to in the literature as neuronal apoptosis inhibitory protein (Naip).
Birc1 sequences are publicly available. For example, GenBank Accession Nos: NM—004536 and NP—004527 disclose human Birc1 nucleic acid and protein sequences, respectively and GenBank Accession Nos: NM—010870 and NP—035000 disclose mouse Birc1 nucleic acid and proteins sequences, respectively.
In one example, a Birc1 sequence includes a full-length wild-type (or native) sequence, as well as Birc1 allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to decrease or inhibit caspase biological activity. In certain examples, Birc1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a native Birc1. In other examples, Birc1 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. NM—010870 or NM—004536, and retains Birc1 activity.
Binding or stable binding: An association between two substances or molecules, such as the hybridization of one nucleic acid molecule to another (or itself), the association of an antibody with a peptide, or the association of a protein with another protein or nucleic acid molecule. An oligonucleotide molecule binds or stably binds to a target nucleic acid molecule if a sufficient amount of the oligonucleotide molecule forms base pairs or is hybridized to its target nucleic acid molecule, to permit detection of that binding.
Binding can be detected by any procedure known to one skilled in the art, such as by physical or functional properties of the target: oligonucleotide complex. For example, binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like.
Physical methods of detecting the binding of complementary strands of nucleic acid molecules, include but are not limited to, such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. For example, one method involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt. In another example, the method involves detecting a signal, such as a detectable label, present on one or both nucleic acid molecules (or antibody or protein as appropriate).
The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (Tm) at which 50% of the oligomer is melted from its target. A higher (Tm) means a stronger or more stable complex relative to a complex with a lower (Tm).
Bone marrow stromal cell antigen 1 (BST-1): A glycosylphosphatidylinositol (GPI)-anchored protein involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP), as well as activation of white blood cells. Also known in the art as CD157. The term bone marrow stromal cell antigen 1 (BST-1) includes any BST-1 gene, cDNA, mRNA, or protein from any organism and that is a BST-1 that has BST-1 biological activity. BST-1 sequences are publicly available. For example, GenBank Accession Nos: BT019502 and AAV38309 disclose human BST-1 nucleic acid and proteins sequences, respectively.
In one example, a BST-1 sequence includes a full-length wild-type (or native) sequence, as well as BST-1 allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to function in adhesion and chemotaxis. In certain examples, BST-1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a native BST-1. In other examples, BST-1 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. BT019502, and retains BST-1 activity.
CD163: A hemoglobin scavenger receptor. The term CD163 includes any CD163 gene, cDNA, mRNA, or protein from any organism and that is a CD163 that can function as a hemoglobin scavenger receptor. CD163 sequences are publicly available. For example, GenBank Accession Nos: Y18388 and CAB45233 disclose human CD163 nucleic acid and protein sequences, respectively and GenBank Accession Nos: NM—053094 and NP—444324 disclose mouse CD163 nucleic acid and proteins sequences, respectively.
In one example, a CD163 sequence includes a full-length wild-type (or native) sequence, as well as CD163 allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to function as a hemoglobin scavenger receptor. In certain examples, CD163 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a native CD163. In other examples, CD163 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. Y18388 or NM—053094, and retains CD163 activity.
cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences which determine transcription. cDNA can be synthesized by reverse transcription from messenger RNA extracted from cells.
Clinical indications of stroke: One or more signs or symptoms that are associated with a subject having (or had) a stroke, such as an ischemic stroke. Particular examples include, but are not limited to: headache, sensory loss (such as numbness, particularly confined to one side of the body or face), paralysis (such as hemiparesis), pupillary changes, blindness (including bilateral blindness), ataxia, memory impairment, dysarthria, somnolence, and other effects on the central nervous system recognized by those of skill in the art.
Complementarity and percentage complementarity: Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide molecule remains detectably bound to a target nucleic acid sequence under the required conditions.
Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, that is, the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.
In the present disclosure, “sufficient complementarity” means that a sufficient number of base pairs exist between an oligonucleotide molecule and a target nucleic acid sequence (such as an ischemic stroke-related sequence, for example any of the sequences listed in Tables 2-5) to achieve detectable binding. When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In general, sufficient complementarity is at least about 50%, for example at least about 75% complementarity, at least about 90% complementarity, at least about 95% complementarity, at least about 98% complementarity, or even at least about 100% complementarity.
A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate oligonucleotides for use under the desired conditions is provided by Beltz et al. Methods Enzymol. 100:266-285, 1983, and by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
DNA (deoxyribonucleic acid): A long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides, referred to as codons, in DNA molecules code for amino acid in a polypeptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
Deletion: The removal of one or more nucleotides from a nucleic acid sequence (or one or more amino acids from a protein sequence), the regions on either side of the removed sequence being joined together.
Differential expression: A difference, such as an increase or decrease, in the conversion of the information encoded in a gene (such as an ischemic stroke related gene) into messenger RNA, the conversion of mRNA to a protein, or both. In some examples, the difference is relative to a control or reference value, such as an amount of gene expression that is expected in a subject who has not had an ischemic stroke or an amount expected in a subject who has had an ischemic stroke. Detecting differential expression can include measuring a change in gene expression.
Downregulated or inactivation: When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in a decrease in production of a gene product. A gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, gene downregulation or deactivation includes processes that decrease transcription of a gene or translation of mRNA.
Examples of processes that decrease transcription include those that facilitate degradation of a transcription initiation complex, those that decrease transcription initiation rate, those that decrease transcription elongation rate, those that decrease processivity of transcription and those that increase transcriptional repression. Gene downregulation can include reduction of expression above an existing level. Examples of processes that decrease translation include those that decrease translational initiation, those that decrease translational elongation and those that decrease mRNA stability.
Gene downregulation includes any detectable decrease in the production of a gene product. In certain examples, production of a gene product decreases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal cell). In one example, a control is a relative amount of gene expression or protein expression in a PBMC in a subject who has not suffered an ischemic stroke.
Evaluating a stroke: To determine whether an ischemic stroke has occurred in a subject, to determine the severity of an ischemic stroke, to determine the likely neurological recovery of a subject who has had an ischemic stroke, or combinations thereof.
Expression: The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.
The expression of a nucleic acid molecule can be altered relative to a normal (wild type) nucleic acid molecule. Alterations in gene expression, such as differential expression, includes but is not limited to: (1) overexpression; (2) underexpression; or (3) suppression of expression. Alternations in the expression of a nucleic acid molecule can be associated with, and in fact cause, a change in expression of the corresponding protein.
Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (wild type) situation. This includes but is not necessarily limited to: (1) a mutation in the protein such that one or more of the amino acid residues is different; (2) a short deletion or addition of one or a few (such as no more than 10-20) amino acid residues to the sequence of the protein; (3) a longer deletion or addition of amino acid residues (such as at least 20 residues), such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount of the protein compared to a control or standard amount; (5) expression of a decreased amount of the protein compared to a control or standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporally regulated expression of the protein (such that the protein is expressed when it normally would not be, or alternatively is not expressed when it normally would be); (8) alteration in stability of a protein through increased longevity in the time that the protein remains localized in a cell; and (9) alteration of the localized (such as organ or tissue specific or subcellular localization) expression of the protein (such that the protein is not expressed where it would normally be expressed or is expressed where it normally would not be expressed), each compared to a control or standard. Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who has not had an ischemic stroke) as well as laboratory values, even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.
Laboratory standards and values may be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.
Fc fragment of IgG, high affinity Ia, receptor for (high affinity immunoglobulin G receptor Fc gamma RI, FcγRI): One of three classes of receptors for the Fc fragment of IgG (FcγR) that participates in immune complex clearance. Binding of ligand to FcγRI initiates multiple immune activation events, such as phagocytosis, expression of proinflammatory cytokines, and cytotoxicity against Ig-coated target cells. Also known in the art as CD64. The term FcγRI includes any FcγRI gene, cDNA, mRNA, or protein from any organism and that is a FcγRI that can function in immune complex clearance. FcγRI sequences are publicly available. For example, GenBank Accession Nos: NM—000566 (nucleic acid) and CAI12557 (protein) and NP—000557 (protein) disclose human FcγRI sequences.
In one example, a FcγRI sequence includes a full-length wild-type (or native) sequence, as well as FcγRI allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to function in immune complex clearance. In certain examples, FcγRI has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a native FcγRI. In other examples, FcγRI has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. NM—000566 and retains FcγRI activity.
Gene expression profile (or fingerprint): Differential or altered gene expression can be detected by changes in the detectable amount of gene expression (such as cDNA or mRNA) or by changes in the detectable amount of proteins expressed by those genes. A distinct or identifiable pattern of gene expression, for instance a pattern of high and low expression of a defined set of genes or gene-indicative nucleic acids such as ESTs; in some examples, as few as one or two genes provides a profile, but more genes can be used in a profile, for example at least 3, at least 4, at least 5, at least 10, at least 20, at least 25, at least 50, at least 80, at least 100, at least 190, at least 200, at least 300, at least 400, at least 500, at least 550, at least 600, or at least 630 or more. A gene expression profile (also referred to as a fingerprint) can be linked to a tissue or cell type (such as PBMCs), to a particular stage of normal tissue growth or disease progression (such as ischemic stroke), or to any other distinct or identifiable condition that influences gene expression in a predictable way. Gene expression profiles can include relative as well as absolute expression levels of specific genes, and can be viewed in the context of a test sample compared to a baseline or control sample profile (such as a sample from a subject who has not had an ischemic stroke). In one example, a gene expression profile in a subject is read on an array (such as a nucleic acid or protein array).
Hybridization: To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). The following is an exemplary set of hybridization conditions and is not limiting:
Very High Stringency (Detects Sequences that Share 90% Identity)
Hybridization: 5×SSC at 65° C. for 16 hours
Wash twice: 2×SSC at room temperature (RT) for 15 minutes each
Wash twice: 0.5×SSC at 65° C. for 20 minutes each
High Stringency (Detects Sequences that Share 80% Identity or Greater)
Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours
Wash twice: 2×SSC at RT for 5-20 minutes each
Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each
Low Stringency (Detects Sequences that Share Greater than 50% Identity)
Hybridization: 6×SSC at RT to 55° C. for 16-20 hours
Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.
Hypothetical protein FLJ22662 Laminin A motif: The term hypothetical protein FLJ22662 Laminin A motif sequence includes any hypothetical protein FLJ22662 Laminin A motif sequence gene, cDNA, mRNA, or protein from any organism and that is a hypothetical protein FLJ22662 Laminin A motif sequence. In particular examples, hypothetical protein FLJ22662 Laminin A motif is part of a basement membrane.
Hypothetical protein FLJ22662 Laminin A motif sequences are publicly available. For example, GenBank Accession Nos: BC063561 (nucleic acid), BC000909 (nucleic acid), AAH00909 (protein) and AAH63561 (protein) disclose human sequences. In one example, a hypothetical protein FLJ22662 Laminin A motif sequence includes a full-length wild-type (or native) sequence, as well as hypothetical protein FLJ22662 Laminin A motif allelic variants, variants, fragments, homologs or fusion sequences. In certain examples, CD163 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a native hypothetical protein FLJ22662 Laminin A motif. In other examples, hypothetical protein FLJ22662 Laminin A motif has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. BC063561 or BC000909, and retains FLJ22662 activity.
Insertion: The addition of one or more nucleotides to a nucleic acid sequence, or the addition of one or more amino acids to a protein sequence.
Ischemic stroke: An ischemic stroke occurs when a blood vessel that supplies blood to the brain is blocked or narrowed (as contrasted with a hemorrhagic stroke which develops when an artery in the brain leaks or ruptures and causes bleeding inside the brain tissue or near the surface of the brain). The blockage can be a blood clot that forms or lodges inside the blood vessel (thrombus) or an object (such as an air bubble or piece of tissue) that moves through the blood from another part of the body (embolus).
Ischemic Stroke-related (or associated) molecule: A molecule whose expression is affected by an ischemic stroke. Such molecules include, for instance, nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins. Specific examples include those listed in Tables 2-5, as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby) whose expression is altered (such as upregulated or downregulated) in response to an ischemic stroke.
Examples of ischemic stroke-related molecules whose expression is upregulated following an ischemic stroke include sequences involved in white blood cell activation and differentiation, sequences related to hypoxia, sequences involved in vascular repair, and sequences related to a specific PBMC response to the altered cerebral microenvironment, such as those genes listed in Table 5. Specific examples of ischemic stroke-related molecules whose expression is upregulated following an ischemic stroke include CD163; hypothetical protein FLJ22662 Laminin A motif; bone marrow stromal cell antigen 1 (also known as CD157); Fc fragment of IgG, high affinity Ia, receptor for (CD64); baculoviral IAP repeat-containing protein 1 (also known as neuronal apoptosis inhibitory protein); and KIAA0146, or any one of these.
Ischemic stroke-related molecules can be involved in or influenced by an ischemic stroke in different ways, including causative (in that a change in an ischemic stroke-related molecule leads to development of or progression to an ischemic stroke) or resultive (in that development of or progression to an ischemic stroke causes or results in a change in the ischemic stroke-related molecule).
Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been “isolated” include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. For example, an isolated cell, such as an isolated PBMC is one that is substantially separated from other cells, such as other blood cells.
KIAA0146: The term KIAA0146 includes any KIAA0146 gene, cDNA, mRNA, or protein from any organism and that is a KIAA0146 sequence. KIAA0146 sequences are publicly available. For example, GenBank Accession Nos: AAH15561 (protein), BAA09767 (protein), D63480 (nucleic acid), and BC015561 (nucleic acid) disclose human KIAA0146 sequences.
In one example, a KIAA0146 sequence includes a full-length wild-type (or native) sequence, as well as KIAA0146 allelic variants, variants, fragments, homologs or fusion sequences. In certain examples, KIAA0146 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a native KIAA0146. In other examples, KIAA0146 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. D63480 or BC015561, and retains KIAA0146 activity.
Label: An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
Neurological sequelae: Any abnormality of the nervous system (such as the central nervous system) following or resulting from a disease or injury or treatment, for example following an ischemic stroke.
Nucleic acid array: An arrangement of nucleic acids (such as DNA or RNA) in assigned locations on a matrix, such as that found in cDNA arrays, or oligonucleotide arrays.
Nucleic acid molecules representing genes: Any nucleic acid, for example DNA (intron or exon or both), cDNA, or RNA (such as mRNA), of any length suitable for use as a probe or other indicator molecule, and that is informative about the corresponding gene.
Nucleic acid molecules: A deoxyribonucleotide or ribonucleotide polymer including, without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA. The nucleic acid molecule can be double-stranded or single-stranded. Where single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. In addition, nucleic acid molecule can be circular or linear.
The disclosure includes isolated nucleic acid molecules that include specified lengths of an ischemic stroke-related nucleotide sequence, for example those listed in Tables 2-5. Such molecules can include at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 consecutive nucleotides of these sequences or more, and can be obtained from any region of an ischemic stroke-related nucleic acid molecule.
Nucleotide: Includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.
Oligonucleotide: A plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.
Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 nucleotides, for example at least 8, at least 10, at least 15, at least 20, at least 21, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100 or even at least 200 nucleotides long, or from about 6 to about 50 nucleotides, for example about 10-25 nucleotides, such as 12, 15 or 20 nucleotides.
Oligonucleotide probe: A short sequence of nucleotides, such as at least 8, at least 10, at least 15, at least 20, at least 21, at least 25, or at least 30 nucleotides in length, used to detect the presence of a complementary sequence by molecular hybridization. In particular examples, oligonucleotide probes include a label that permits detection of oligonucleotide probe:target sequence hybridization complexes.
Open reading frame (ORF): A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.
Peripheral blood mononuclear cells (PBMCs): Cells present in the blood that have one round nucleus. Examples include lymphocytes, monocytes, and natural killer cells. PBMCs do not include neutrophils, eosinophils or basophils.
Primers: Short nucleic acid molecules, for instance DNA oligonucleotides 10-100 nucleotides in length, such as about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand. Primer pairs can be used for amplification of a nucleic acid sequence, such as by PCR or other nucleic acid amplification methods known in the art.
Methods for preparing and using nucleic acid primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ©1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). One of ordinary skill in the art will appreciate that the specificity of a particular primer increases with its length. Thus, for example, a primer including 30 consecutive nucleotides of an ischemic stroke-related nucleotide molecule will anneal to a target sequence, such as another homolog of the designated ischemic stroke-related protein, with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, primers can be selected that include at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of a ischemic stroke-related nucleotide sequence. Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell. For example, a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation. Similarly, a purified oligonucleotide preparation is one in which the oligonucleotide is more pure than in an environment including a complex mixture of oligonucleotides. In addition, a purified cell, such as a purified PBMC, is one that is substantially separated from other cells, such as other blood cells. In one example, purified PBMCs are at least 90% pure, such as at least 95% pure, or even at least 99% pure.
Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.
Sample: A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. In one example, a sample includes peripheral blood mononuclear cells (PBMCs).
Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options can be set as follows: −i is set to a file containing the first nucleic acid sequence to be compared (such as C:\seq1.txt); −j is set to a file containing the second nucleic acid sequence to be compared (such as C:\seq2.txt); −p is set to blastn; −o is set to any desired file name (such as C:\output.txt); −q is set to 1; −r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq−i c:\seq1.txt−j c:\seq2.txt−p blastn−o c:\output.txt−q−1−r2.
To compare two amino acid sequences, the options of B12seq can be set as follows: −i is set to a file containing the first amino acid sequence to be compared (such as C:\seq1.txt); −j is set to a file containing the second amino acid sequence to be compared (such as C:\seq2.txt); −p is set to blastp; −o is set to any desired file name (such as C:\output.txt); and all other options are left at their default setting.
For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq−i c:\seq1.txt−j c:\seq2.txt−p blastp−o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1154 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15≧20*100=75).
For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.
When aligning short peptides (fewer than around 30 amino acids), the alignment is be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.
One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity determined by this method. An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
One of skill in the art will appreciate that the particular sequence identity ranges are provided for guidance only; it is possible that strongly significant homologs could be obtained that fall outside the ranges provided.
Sequences involved in (or related to) white blood cell activation and differentiation: Nucleic acid molecules (such as mRNA, cDNA, gene) and the corresponding protein, whose expression is altered (such as upregulated or downregulated) in connection with the specialization, mobilization, or proliferation of white blood cells, or combinations thereof, for example sequences that are differentially expressed to cause (or are differentially expressed as a result of) specialization, mobilization, and/or proliferation of white blood cells.
Exemplary sequences involved in white blood cell activation and differentiation include genes involved in cell adhesion, enzymes involved in the cell membrane remodeling allowing preparation for change to a more differentiated state, and genes related to cell-cell interactions. Particular examples include, but are not limited to, CD163; hypothetical protein FLJ22662 Laminin A motif; amyloid beta (A4) precursor-like protein 2; N-acetylneuraminate pyruvate lysase; v-fos FBJ murine osteosarcoma viral oncogene homolog; toll-like receptor 2; chondroitin sulfate proteoglycan 2 (versican); interleukin 13 receptor, alpha 1; CD14 antigen; bone marrow stromal cell antigen 1 (also known as CD157); complement component 1, q subcomponent, receptor 1; and paired immunoglobin-like type 2 receptor alpha; and Fc fragment of IgG, high affinity Ia, receptor for (CD64).
Sequences involved in (or related to) hypoxia: Nucleic acid molecules (such as mRNA, cDNA, gene) and the corresponding protein, whose expression is altered (such as upregulated or downregulated) in response to decreased available oxygen in the blood and tissues. For example, the brain is hypoxic following an ischemic stroke. Particular examples include, but are not limited to, adrenomedullin; dual specificity phosphatase 1; cytochrome b-245, beta polypeptide (chronic granulomatous disease); ;eukotriene A4 hydrolase; erythroblastosis virus E26 oncogene homolog 2 (avian); and neutrophil cytosolic factor 2 (65 kDa, chronic granulomatous disease, autosomal 2).
Sequences involved in (or related to) vascular repair: Nucleic acid molecules (such as mRNA, cDNA, gene) and the corresponding protein, whose expression is altered (such as upregulated or downregulated) in response to injury to a blood vessel. Particular examples include, but are not limited to, thrombomodulin; ectonucleoside triphosphate diphosphohydrolase 1; and CD36 antigen (collagen type I receptor, thrombospondin receptor).
Sequences involved in (or related to) a specific PBMC response to the altered cerebral microenvironment: Nucleic acid molecules (such as mRNA, cDNA, gene) and the corresponding protein, whose expression is altered (such as upregulated or downregulated) in PBMCs in response to changes in the brain microenvironment.
Examples include those potentially associated with enhanced neurotransmitter degradation (such as catechol-o-methyl transferase and glutamine ligase), those that permit increased modulation of Ca2+ homeostasis in the cerebral environment, genes involved in the inhibition of neuronal apoptosis (such as the neuronal apoptosis inhibitory protein and Ets2), genes involved in proNGF-induced neuronal cell death (such as sortilin), genes involved in apoptotic cell death in the hippocampus after global cerebral ischemic injury (such as phospholipid scramblase 1), and genes involved in neurite growth in neuronal development (such as growth arrest-specific 7).
Particular examples include, but are not limited to, catechol-O-methyltransferase; glutamate-ammonia ligase (glutamine ligase); 5100 calcium binding protein A8 (calgranulin A); neuronal apoptosis inhibitory protein: Homo sapiens transcribed sequence with strong similarity to protein sp:Q13075 (H. sapiens) BIR1_HUMAN Baculoviral IAP repeat-containing protein 1; sortilin; phospholipid scramblase 1; growth-arrest-specific 7; and GLI pathogenesis-related 1 (glioma).
Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.
Target sequence: A sequence of nucleotides located in a particular region in the human genome that corresponds to a desired sequence, such as ischemic stroke related sequence. The target can be for instance a coding sequence; it can also be the non-coding strand that corresponds to a coding sequence. Examples of target sequences include those sequences associated with ischemic stroke, such as any of those listed in Tables 2-5.
Test agent: Any substance, including, but not limited to, a protein (such as an antibody), nucleic acid molecule, organic compound, inorganic compound, or other molecule of interest. In particular examples, a test agent can permeate a cell membrane (alone or in the presence of a carrier).
Therapeutically effective amount: An amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response. A therapeutic agent, such as an anticoagulant or a thrombolytic agent, is administered in therapeutically effective amounts.
Effective amounts a therapeutic agent can be determined in many different ways, such as assaying for a reduction in atherosclerotic disease or improvement of physiological condition of a subject having vascular disease. Effective amounts also can be determined through various in vitro, in vivo or in situ assays.
Therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.
In one example, it is an amount sufficient to partially or completely alleviate symptoms of ischemic stroke within a subject. Treatment can involve only slowing the progression of the ischemic stroke temporarily, but can also include halting or reversing the progression of the ischemic stroke permanently. For example, a pharmaceutical preparation can decrease one or more symptoms of ischemic stroke, for example decrease a symptom by at least 20%, at least 50%, at least 70%, at least 90%, at least 98%, or even at least 100%, as compared to an amount in the absence of the pharmaceutical preparation.
Thrombolytics: Agents that promote lysis of thrombi that occlude a cerebral vessel. Examples include, but are not limited to, tissue plasminogen activator (tPA), urokinase, and pro-urokinase. Administration of antithrombotics is one treatment for ischemic stroke, and is often a first line treatment for ischemic stroke. For example, intravenous t-PA can be administered within 3 hours of ischemic stroke onset. Intra-arterial thrombolytic therapy and mechanical clot-retrieval devices can be used to promote rapid lysis of thrombi.
Treating a disease: “Treatment” refers to a therapeutic intervetnion that ameliorates a sign or symptom of a disease or pathological condition, such a sign or symptom of vascular disease. Treatment can also induce remission or cure of a condition, such as an ischemic stroke. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease, such as preventing development of a disease or disorder that results from an ischemic stroke. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient.
Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity.
In one example, includes culturing cells (such as PBMCs) under conditions sufficient to mimic an ischemic stroke, such as culturing the cells under hypoxic conditions, hypoglycemic conditions, or both.
In another example, includes administering a test agent to a cell culture or a subject sufficient to allow the desired activity. In particular examples, the desired activity is altering the activity (such as the expression) of an ischemic stroke-related molecule.
Upregulated or activation: When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in an increase in production of a gene product. A gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, gene upregulation or activation includes processes that increase transcription of a gene or translation of mRNA.
Examples of processes that increase transcription include those that facilitate formation of a transcription initiation complex, those that increase transcription initiation rate, those that increase transcription elongation rate, those that increase processivity of transcription and those that relieve transcriptional repression (for example by blocking the binding of a transcriptional repressor). Gene upregulation can include inhibition of repression as well as stimulation of expression above an existing level. Examples of processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability.
Gene upregulation includes any detectable increase in the production of a gene product. In certain examples, production of a gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal cell). In one example, a control is a relative amount of gene expression in a PBMC in a subject who has not suffered an ischemic stroke.
The inventors have identified 22-637 genes whose expression is altered (such as upregulated or downregulated) following an ischemic stroke. The number of genes identified depended on the specificity and sensitivity of the algorithm used. For example, using the PAM dataset 22 ischemic stroke related genes were identified (Table 5), using the Westfall and Young dataset 82 ischemic stroke related genes were identified (Table 4), using the Bonferroni correction set 190 ischemic stroke related genes were identified (Table 3), and using the Benjamini & Yekutieli set 637 ischemic stroke related genes were identified (Table 2). One skilled in the art will appreciate that changes in protein expression can be detected as an alternative to detecting gene expression.
Several genes not previously associated with ischemic stroke were identified, such as at least CD163; hypothetical protein FLJ22662 Laminin A motif; bone marrow stromal cell antigen 1 (BST-1, also referred to in the literature as CD157); Fc fragment of IgG, high affinity Ia, receptor for (FcγRI, also known as CD64); baculoviral IAP repeat-containing protein 1; and KIAA0146. In particular examples, all of these genes were upregulated following an ischemic stroke. In one example, four classes of genes whose expression was upregulated following an ischemic stroke were identified: sequences involved in activation and differentiation of white blood cells, sequences related to hypoxia, sequences involved in vascular repair, and sequences related to altered cerebral microenvironment. Particular examples of such genes (and their corresponding proteins) are provided in Table 5.
Based on the identification of these ischemic stroke-related molecules, methods were developed to evaluate a stroke. For example, the disclosed methods can be used to diagnose an ischemic stroke, determine the severity of an ischemic stroke, determine the likely neurological recovery of a subject who had an ischemic stroke, or combinations thereof. The method can further include determining an appropriate therapy for a subject found to have experienced an ischemic stroke using the disclosed assays.
The disclosed methods provide a rapid, straightforward, and accurate genetic screening method performed in one assay for evaluating ischemic stroke. It allows identification of subjects who may require anticoagulant therapy following an ischemic stroke. For example, by establishing that an individual has had an ischemic stroke, effective therapeutic measures, such as the emergent administration of a thrombolytic agent or of treatments to prevent stroke recurrence and extension, can be instituted.
Provided herein are methods of evaluating a stroke. Particular examples of evaluating a stroke include determining whether a subject, such as an otherwise healthy subject, or a subject suspected or at risk of having an ischemic stroke, has had an ischemic stroke, assessing the severity of an ischemic stroke, predicting the likelihood of neurological recovery of a subject who has had an ischemic stroke, or combinations thereof. The identification of a subject who has had an ischemic stroke can help to evaluate other clinical data (such as neurological impairment or brain imaging information) to determine whether an ischemic stroke has occurred. In particular examples, the method can determine with a reasonable amount of sensitivity and specificity whether a subject has suffered an ischemic stroke within the previous 72 hours, such as within the previous 48 hours, previous 24 hours, or previous 12 hours. In some examples, isolated or purified PBMCs obtained from the subject are used to determine whether a subject has had an ischemic stroke.
In particular examples, the method also includes administering an appropriate treatment therapy for subjects who have had an ischemic stroke. For example, subjects identified or evaluated as having had an ischemic stroke can then be provided with appropriate treatments, such as anti-platelet agents (for example aspirin) that would be appropriate for a subject identified as having had an ischemic stroke but not as appropriate for a subject who has had a hemorrhagic stroke. It is helpful to be able to classify a subject as having had an ischemic stroke, because the treatments for ischemic stroke are often distinct from the treatments for hemorrhagic stroke. In fact, treating a hemorrhagic stroke with a therapy designed for an ischemic stroke (such as a thrombolytic agent) can have devastating clinical consequences. Hence using the results of the disclosed assays to help distinguish ischemic from hemorrhagic stroke offers a substantial clinical benefit, and allows subjects to be selected for treatments appropriate to ischemic stroke but not hemorrhagic stroke.
In particular examples, methods of evaluating a stroke involve detecting differential expression (such as an increase or decrease in gene or protein expression) in any combination of at least four ischemic stroke-related molecules of the subject, such as any combination of at least four of the genes (or proteins) listed in any of Tables 2-5. In one example, the method includes screening expression of one or more of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, or a combination of ischemic stroke-related molecules that includes at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 of these molecules. For example, the method can include screening expression of CD163, along with other ischemic stroke-related molecules (such as any combination that includes at least 3 additional molecules listed in Tables 2-5).
Differential expression can be represented by increased or decreased expression in the at least one ischemic stroke-related molecule (for instance, a nucleic acid or a protein). For example, differential expression includes, but is not limited to, an increase or decrease in an amount of a nucleic acid molecule or protein, the stability of a nucleic acid molecule or protein, the localization of a nucleic acid molecule or protein, or the biological activity of a nucleic acid molecule or protein. Specific examples include evaulative methods in which changes in gene expression in at least four ischemic stroke-related nucleic acid molecules (or corresponding protein) are detected (for example nucleic acids or proteins obtained from a subject thought to have had or known to have had an ischemic stroke), such as changes in gene (or protein) expression in any combination of at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 100, at least 150, at least 160, at least 170, at least 175, at least 180, at least 185, at least 200, at least 250, at least 300, at least 400, at least 500, at least 510, at least 550, at least 575, at least 600, at least 620, at least 630, or at least 637 ischemic stroke-related molecules. Exemplary ischemic stroke-related molecules are provided in Tables 2-5.
In particular examples a change in expression is detected in a subset of ischemic stroke-related molecules (such as nucleic acid sequences or protein sequences) that selectively evaluate a stroke, for example to determine if a subject has had an ischemic stroke. In a particular example, the subset of molecules can include a set of any combination of four ischemic stroke-related genes listed in Table 5, or a set of any combination of 22 ischemic stroke-related genes listed in Table 5. In a particular example, the subset of molecules includes any combination of at least one gene (or protein) from each class of the four classes listed in Table 5, such as at least 2, at least 3, at least 5, or at least 10 genes from each class listed in Table 5.
In a particular example, differential expression is detected in ischemic stroke-related molecules that are both upregulated and down regulated. For example, increased expression of one or more of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, and decreased gene (or protein) expression of one or more of intercellular adhesion molecule 2, protein kinase D2, GATA binding protein 3, hypothetical protein FLJ20257, or protein kinase C, theta, indicates that the subject has had a stroke, has had a severe stroke, has a lower likelihood of neurological recovery, or combinations thereof. For example, differential expression can be detected by determining if the subject has increased gene (or protein) expression of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, and determining if the subject has decreased gene (or protein) expression of intercellular adhesion molecule 2, protein kinase D2, GATA binding protein 3, hypothetical protein FLJ20257, and protein kinase C, theta.
In particular examples, the number of ischemic stroke-related genes screened is at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 100, at least 150, at least 160, at least 170, at least 175, at least 180, at least 185, at least 200, at least 250, at least 300, at least 400, at least 500, at least 510, at least 550, at least 575, at least 600, at least 620, at least 630, or at least 637 ischemic stroke-related molecules. In other examples, the methods employ screening no more than 637, no more than 630, no more than 620, no more than 600, no more than 575, no more than 550, no more than 510, no more than 500, no more than 400, no more than 300, no more than 250, no more than 200, no more than 185, no more than 180, no more than 175, no more than 170, no more than 160, no more than 150, no more than 100, no more than 50, no more than 25, no more than 20, no more than 15, no more than 10, no more than 5, or no more than 4 ischemic stroke-related genes. Examples of particular ischemic stroke-related genes are shown in Tables 2-5. In one example, the number of ischemic stroke-related genes screened includes at least one gene from each class listed in Table 5, such as at least 2, at least 3, at least 5, or at least 10 genes from each class listed in Table 5.
In certain methods, differential expression includes over- or under-expression of an ischemic stroke-related molecule. For instance, differential expression can include overexpression, for instance overexpression of any combination of at least 4 molecules (such at least 10 or at least 20 molecules) shown in Table 5, or any combination of at least 4 molecules in any of Tables 2-4 with a positive t-statistic value, such as a t-statistic value of at least 3, such as at least 3.5, at least 3.6 or even at least 3.7. In a particular example, differential expression includes overexpression of any combination of at least one gene from each class listed in Table 5, such as at least 2, at least 3, at least 5, or at least 10 genes from each of the classes listed in Table 5. In another example, differential expression includes underexpression, for instance underexpression of any combination of at least 5 molecules (such at least 50 or at least 150 molecules) shown in Tables 2-4 with a negative t-statistic value, such as a t-statistic value of less than −3, such as less than −3.5, less than -3.6 or even less than −3.7. In a specific example, differential expression includes any combination of underexpression or overexpression of at least 4 ischemic stroke-related molecules shown in Tables 2-4, such as overexpression of at least 3 ischemic stroke-related molecules shown in Tables 2-5 with a positive t-statistic value and underexpression of at least one ischemic stroke related molecule shown in Tables 2-4 with a negative t-statistic value, or for example overexpression of at least 4 ischemic stroke-related molecules shown in Tables 2-5 with a positive t-statistic value, or for example, overexpression of at least 2 ischemic stroke-related molecules shown in Tables 2-5 with a positive t-statistic value and underexpression of at least 2 ischemic stroke related molecules shown in Tables 2-4 with a negative t-statistic value.
In some examples, differential expression of proteins that are associated with ischemic stroke includes detecting patterns of such expression, such as detecting upregulation of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, detecting downregulation of intercellular adhesion molecule 2, protein kinase D2, GATA binding protein 3, hypothetical protein FLJ20257, or protein kinase C, theta, or combinations thereof. For example, detecting upregulation or downregulation can include a magnitude of change of at least 25%, at least 50%, at least 100%, or even at least 200%, such as a magnitude of change of at least 25% for CD163; at least 25% for hypothetical protein FLJ22662 Laminin A motif; at least 25% for BST-1; FcγRI; at least 25% for baculoviral IAP repeat-containing protein 1; at least 25% for KIAA0146; at least 25% for intercellular adhesion molecule 2; at least 25% for protein kinase D2; at least 25% for GATA binding protein 3; at least 25% for hypothetical protein FLJ20257; and at least 25% for protein kinase C, theta. Alternatively, upregulation is detected by a level having a t-value of at least 3 and downregulation is detected by a level having a t-value value of no more than −3.
In particular examples, the disclosed method of evaluating a stroke is at least 78% sensitive (such as at least 80% sensitive, at least 85% sensitive, at least 90% sensitive, or at least 95% sensitive) and at least 80% specific (such as at least 85% specific, at least 90% specific, at least 95% specific, or at least 98% specific) for determining whether a subject has had an ischemic stroke.
As used herein, the term “ischemic stroke-related molecule” includes ischemic stroke-related nucleic acid molecules (such as DNA, RNA, for example cDNA or mRNA) and ischemic stroke-related proteins. The term is not limited to those molecules listed in Tables 2-5 (and molecules that correspond to those listed), but also includes other nucleic acid molecules and proteins that are influenced (such as to level, activity, localization) by or during an ischemic stroke, including all of such molecules listed herein. Examples of particular ischemic stroke-related genes are listed in Tables 2-5, such as CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146. In examples where the ischemia-related molecule is an ischemia -related nucleic acid sequence, methods of detecting differential expression can include in vitro nucleic acid amplification, nucleic acid hybridization (which can include quantified hybridization), RT-PCR, real time PCR, or combinations thereof. In examples where the ischemia-related molecule is an ischemia -related protein sequence, methods of detecting differential expression can include in vitro hybridization (which can include quantified hybridization) such as hybridization to a protein-specific binding agent for example an antibody, quantitative spectroscopic methods (for example mass spectrometry, such as surface-enhanced laser desorption/ionization (SELDI)-based mass spectrometry) or combinations thereof.
In particular examples, methods of evaluating a subject who has had or is thought to have had an ischemic stroke includes determining a level of expression (for example in a PBMC) of any combination of at least 4 of the genes (or proteins) listed in Tables 2-5, such as at least 10, at least 15, at least 20, or at least 22 of the genes listed in Table 5, such as at least 150, at least 180, or at least 185 of the gene listed in Table 3, or any combination of at least 500, at least 600, or at least 630 of the genes listed in Table 2. In one example, the method includes determining a level of expression of at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146, or any combination of ischemic stroke related molecules that includes 1, 2, 3, 4, 5, or 6 of these molecules. In one example, the method includes determining a level of expression of at least one gene from each class listed in Table 5, such as at least 2, at least 3, at least 5, or at least 10 genes from each class.
Methods of evaluating a stroke can include diagnosing a stroke, stratifying the seriousness of a cerebral ischemic event, and predicting neurological recovery. Similarly, methods of evaluating a stroke can include determining the severity of a stroke, predicting neurological recovery, or combinations thereof. For example, a change in expression in any combination of at least 4 of the genes listed in Tables 2-5 indicates that the subject has had an ischemic stroke. For example, an increase in expression in one or more of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146 in particular examples indicates that the subject has had an ischemic stroke.
Determining the level of expression can involve measuring an amount of the ischemia-related molecules in a sample derived from the subject, such as a purified PBMC sample. Such an amount can be compared to that present in a control sample (such as a sample derived from a subject who has not had an ischemic stroke or a standard ischemic stroke-related molecule level in analogous samples from a subject not having ischemia or not having a predisposition developing ischemia), wherein a difference (such as an increase or a decrease reflecting an upregulation or downregulation, respectively) in the level of any combination of at least 4 ischemia-related molecules listed in Tables 2-5, such as any combination of at least 4 ischemia-related molecules listed in Table 5, in the subject relative to the control sample is diagnostic for ischemic stroke.
In other examples, the method includes determining a level of expression of any combination of at least four sequences listed in Table 5, such as at least 10, or at least 22 of the sequences listed in Table 5, for example at least 150 of the genes listed in Table 3, such as at least 160, at least 170, at least 175, at least 180, or at least 185 of the genes listed in Table 3, or at least 500 of the ischemic stroke-related molecules listed in Table 2, such as at least 600 of the ischemic stroke-related molecules listed in Table 2. A change in expression in at least four genes listed in Table 5 (or the corresponding proteins), such as at least 22 of the genes (or the corresponding proteins) listed in Table 5, such as 150 or more of the genes listed in Table 3 (or the corresponding proteins), such as 500 or more of the genes listed in Table 2 (or the corresponding proteins, indicates that the subject has had a more severe stroke, has a higher risk of long term adverse neurological sequelae, or combinations thereof, than a subject having a change in expression in less than 500 of the molecules listed in Table 3, less than 150 of the molecules listed in Table 3 or less than 22 (or less than four) of the molecules listed in Table 5. Determining the level of expression can involve measuring an amount of the ischemia-related molecules in a sample derived from the subject. Such an amount can be compared to that present in a control sample (such as a sample derived from a subject who has not had an ischemic stroke or a sample derived from the subject at an earlier time), wherein a difference (such as an increase or a decrease reflecting an upregulation or downregulation, respectively) in the level of at least 4 or at least 22 of the ischemia-related molecules listed in Table 5 (such as at least 150 of the ischemia-related molecules listed in Table 3 or such as at least 500 of the ischemia-related molecules listed in Table 2) in the subject relative to the control sample indicates that the subject has had a more severe stroke, has a higher risk of long term adverse neurological sequelae, or both.
The disclosed methods can further include administering to the subject an appropriate treatment to avoid or reduce ischemic injury, if the presence of differential expression indicates that the subject has had an ischemic stroke. Since the results of the disclosed assays are reliable predictors of the ischemic nature of the stroke, the results of the assay can be used (alone or in combination with other clinical evidence and brain scans) to determine whether thrombolytic therapy designed to lyse a neurovascular occlusion such as a thrombus (for example by using tissue plasminogen activator or streptokinase) should be administered to the subject. In certain example, thrombolytic therapy is given to the subject once the results of the differential gene assay are known if the assay provides an indication that the stroke is ischemic in nature. Such methods can reduce brain damage following an ischemic stroke.
In particular examples, the method includes determining if there is an alteration in the expression of at least four sequences listed in Table 5, such as at least 10, or at least 22 of the sequences listed in Table 5, for example at least 150 of the genes listed in Table 3, such as at least 160, at least 170, at least 175, at least 180, or at least 185 of the genes listed in Table 3, or at least 500 of the ischemic stroke-related molecules listed in Table 2, such as at least 600 of the ischemic stroke-related molecules listed in Table 2. In some examples, detecting differential expression of at least 4 ischemic stroke-related molecules involves quantitatively or qualitatively analyzing a DNA, mRNA, cDNA, protein, or combinations thereof.
If differenential expression is detected in at least four, at least 22, at least 150, or at least 500 ischemic stroke-related molecules is identified, this indicates that the subject has experienced an ischemic stroke (and not a hemorrhagic stroke), and a treatment is selected to prevent or reduce brain damage or to provide protection from the onset of brain damage. Examples of such treatment include administration of an anticoagulant, an antithrombotic, or combinations thereof. A particular example includes administration of a thrombolytic agent such as t-PA to lyse the blood clot, alone or in combination with one or more agents that prevent further strokes, such as anticoagulants (such as antiplatelet agents), antihypertensive agents, or lipid lowering agents. In particular examples, the level of expression of a protein in a subject can be appropriately increased or decreased by expressing in the subject a recombinant genetic construct that includes a promoter operably linked to a nucleic acid molecule, wherein the nucleic acid molecule includes at least 10 consecutive nucleotides of an ischemic stroke-related nucleic acid sequence (such as any of the sequences listed in Tables 2-5). Expression of the nucleic acid molecule will change expression of the ischemic stroke-related protein. The nucleic acid molecule can be in an antisense orientation relative to the promoter or in sense orientation relative to the promoter. In some examples, the recombinant genetic construct expresses an ssRNA corresponding to an ischemic stroke-related nucleic acid sequence.
In examples of the methods described herein, detecting differential expression of at least four ischemic stroke-related molecules involves determining whether a gene expression profile from the subject indicates development or progression of brain injury.
In particular examples, the disclosed methods are performed following the onset of signs and symptoms associated with ischemic stroke. Examples of such symptoms include, but are not limited to headache, sensory loss (such as numbness, particularly confined to one side of the body or face), paralysis (such as hemiparesis), pupillary changes, blindness (including bilateral blindness), ataxia, memory impairment, dysarthria, somnolence, and other effects on the central nervous system recognized by those of skill in the art. In particular examples, the method of evaluating a stroke is performed after a sufficient period of time for the differential regulation of the genes (or proteins) to occur, for example at least 24 hours after onset of the symptom or constellation of symptoms that have indicated a potential cerebral ischemic event. In other examples, the method is performed prior to performing any diagnostics imaging tests (such as those that can find anatomic evidence of ischemic stroke). For example, it can be difficult for imaging modalities (such as CT and MRI) to detect acute ischemic strokes, at least until brain changes (such as edema) have taken place in response to the ischemia. Hence the assay described herein is able to detect the stroke even before definitive brain imaging evidence of the stroke is known.
The neurological sequelae of an ischemic event in the central nervous system can have consequences that range from the insignificant to devastating, and the disclosed assays permit early and accurate stratification of risk of long-lasting neurological impairment. For example, a test performed as early as within the first 24 hours of onset of signs and symptoms of a stroke, and even as late as 7-14 days or even as late as 90 days or more after the event can provide clinical data that is highly predictive of the eventual care needs of the subject.
The disclosed assay is also able to identify subjects who have had an ischemic stroke in the past, for example more than 2 weeks ago, or even more than 90 days ago. The identification of such subjects helps evaluate other clinical data (such as neurological impairment or brain imaging information) to determine whether an ischemic stroke has occurred.
In particular examples, the disclosed methods provide a lower cost alternative to expensive imaging modalities (such as MRI and CT scans), can be used in instances where those imaging modalities are not available (such as in field hospitals), can be more convenient than placing people in scanners (especially considering that some people are not able to fit in the scanner, or can not be subjected to MRI if they have certain types of metallic implants in their bodies), or combinations thereof.
Appropriate specimens for use with the current disclosure in diagnosing and prognosing ischemic stroke include any conventional clinical samples, for instance blood or blood-fractions (such as serum). Techniques for acquisition of such samples are well known in the art (for example see Schluger et al. J. Exp. Med. 176:1327-33, 1992, for the collection of serum samples). Serum or other blood fractions can be prepared in the conventional manner. For example, about 200 μL of serum can be used for the extraction of DNA for use in amplification reactions. However, if DNA is not amplified, larger amounts of blood can be collected. For example, if at least 5 μg of mRNA is desired, about 20-30 mls of blood can be collected.
In one example, PBMCs are used as a source of isolated nucleic acid molecules or proteins. The inflammatory response from peripheral blood borne white blood cells, in particular monocytes, are also a component of the evolving ischemic lesion (Kochanek et al., Stroke 23:1367-79, 1992). One advantage of using blood (for example instead of brain tissue) is that it is easily available can be drawn serially.
Once a sample has been obtained, the sample can be used directly, concentrated (for example by centrifugation or filtration), purified, amplified, or combinations thereof. For example, rapid DNA preparation can be performed using a commercially available kit (such as the InstaGene Matrix, BioRad, Hercules, Calif.; the NucliSens isolation kit, Organon Teknika, Netherlands. In one example, the DNA preparation method yields a nucleotide preparation that is accessible to, and amenable to, nucleic acid amplification. Similarly, RNA can be prepared using a commercially available kit (such as the RNeasy Mini Kit, Qiagen, Valencia, Calif.).
In particular examples, methods for detecting a change in expression in the disclosed ischemic stroke-related genes listed in Tables 2-5 use the arrays disclosed herein. Arrays can be used to detect the presence of sequences whose expression is upregulated or downregulated in response to an ischemic stroke, such as sequences listed in Tables 2-5, for example using specific oligonucleotide probes or antibody probes. The arrays herein termed “ischemic stroke detection arrays,” are used to evaluate a stroke, for example to determine whether a subject has had an ischemic stroke, determine the severity of the stroke, predict the likelihood of neurological recovery of a subject who has had an ischemic stroke, to identify an appropriate therapy for a subject who has had an ischemic stroke, or combinations thereof. In particular examples, the disclosed arrays can include nucleic acid molecules, such as DNA or RNA molecules, or antibodies.
In one example, the array includes nucleic acid oligonucleotide probes that can hybridize to any combination of at least four of the ischemic stroke-related gene sequences listed in Table 5, at least 150 of the ischemic stroke-related gene sequences listed in Table 3, or at least 500 of the ischemic stroke-related gene sequences listed in Table 2. In particular examples, an array includes oligonucleotides that can recognize all 22 ischemic stroke-associated genes listed in Table 5, all 82 of the ischemic stroke-related gene sequences listed in Table 4, all 190 of the ischemic stroke-related gene sequences listed in Table 3, or all 637 of the ischemic stroke-related gene sequences listed in Table 2. Certain of such arrays (as well as the methods described herein) can include ischemic stroke-related molecules that are not listed in Tables 2-5.
In a specific example, an array includes oligonucleotide probes that can recognize at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, or a probe that can recognize any one of these molecules. For example, the array can include oligonucleotide probes that can recognize at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 of the following, CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146. In another example, the array includes an oligonucleotide probe that can recognize at least CD163, for example in combination with other oligonucleotide probes that recognize other ischemic stroke related molecules (such as any combination of at least 3 of those listed in Tables 2-5).
In another specific example, an array includes oligonucleotide probes that can recognize at least one white blood cell activation and differentiation gene, at least one gene related to hypoxia, at least one gene involved in vascular repair, and at least one gene related to a specific PBMC response to the altered cerebral microenvironment, or at least 2, at least 3, at least 5, or at least 10 genes from each of these families.
In one example, a set of oligonucleotide probes is attached to the surface of a solid support for use in detection of ischemic stroke-associated sequences, such as those nucleic acid sequences (such as cDNA or mRNA) obtained from the subject. Additionally, if an internal control nucleic acid sequence is used (such as a nucleic acid sequence obtained from a PBMC from a subject who has not had an ischemic stroke) an oligonucleotide probe can be included to detect the presence of this control nucleic acid molecule.
The oligonucleotide probes bound to the array can specifically bind sequences obtained from the subject, or amplified from the subject (such as under high stringency conditions). Thus, sequences of use with the method are oligonucleotide probes that recognize ischemic stroke-related sequences, such as gene sequences (or corresponding proteins) listed in Tables 2-5. Such sequences can be determined by examining the sequences of the different species, and choosing oligonucleotide sequences that specifically anneal to a particular ischemic stroke-related sequence (such as those listed in Tables 2-5 or represented by those listed in Tables 2-5), but not others. One of skill in the art can identify other ischemic stroke-associated oligonucleotide molecules that can be attached to the surface of a solid support for the detection of other ischemic stroke-associated nucleic acid sequences.
The methods and apparatus in accordance with the present disclosure takes advantage of the fact that under appropriate conditions oligonucleotides form base-paired duplexes with nucleic acid molecules that have a complementary base sequence. The stability of the duplex is dependent on a number of factors, including the length of the oligonucleotides, the base composition, and the composition of the solution in which hybridization is effected. The effects of base composition on duplex stability can be reduced by carrying out the hybridization in particular solutions, for example in the presence of high concentrations of tertiary or quaternary amines.
The thermal stability of the duplex is also dependent on the degree of sequence similarity between the sequences. By carrying out the hybridization at temperatures close to the anticipated Tm's of the type of duplexes expected to be formed between the target sequences and the oligonucleotides bound to the array, the rate of formation of mis-matched duplexes may be substantially reduced.
The length of each oligonucleotide sequence employed in the array can be selected to optimize binding of target ischemic stroke-associated nucleic acid sequences. An optimum length for use with a particular ischemic stroke-associated nucleic acid sequence under specific screening conditions can be determined empirically. Thus, the length for each individual element of the set of oligonucleotide sequences including in the array can be optimized for screening. In one example, oligonucleotide probes are from about 20 to about 35 nucleotides in length or about 25 to about 40 nucleotides in length.
The oligonucleotide probe sequences forming the array can be directly linked to the support. Alternatively, the oligonucleotide probes can be attached to the support by non-ischemic stroke-associated sequences such as oligonucleotides or other molecules that serve as spacers or linkers to the solid support.
In another example, an array includes protein sequences (or a fragment of such proteins, or antibodies specific to such proteins or protein fragments), which include at least four of the ischemic stroke-related protein sequences listed in Table 5, at least 150 of the ischemic stroke-related protein sequences listed in Table 3, or at least 500 of the ischemic stroke-related protein sequences listed in Table 2. In particular examples, an array includes proteins that can recognize all 22 ischemic stroke-associated proteins listed in Table 5, all 82 of the ischemic stroke-related protein sequences listed in Table 4, all 190 of the ischemic stroke-related proteins listed in Table 3, or all 637 of the ischemic stroke-related proteins listed in Table 2. Such arrays can also contain any particular subset of the sequences listed in Tables 2-5. For example, an array can include probes that can recognize at least one white blood cell activation and differentiation protein, at least one protein related to hypoxia, at least one protein involved in vascular repair, and at least one protein related to a specific PBMC response to the altered cerebral microenvironment, or at least 2, at least 3, at least 5, or at least 10 proteins from each of these families. In another specific example, the array includes probes that recognize one or more of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146. For example, the array can include a probe that recognizes CD163 and additional probes that recognize other ischemic stroke related proteins (such as any combination of at least 3 or at least 22 of those listed in Tables 2-5).
The proteins or antibodies forming the array can be directly linked to the support. Alternatively, the proteins or antibodies can be attached to the support by spacers or linkers to the solid support.
Changes in expression of ischemic stroke-related proteins can be detected using, for instance, an ischemic stroke protein-specific binding agent, which in some instances is labeled with an agent that can be detected. In certain examples, detecting a change in protein expression includes contacting a protein sample obtained from the PBMCs of a subject with an ischemic stroke protein-specific binding agent (which can be for example present on an array); and detecting whether the binding agent is bound by the sample and thereby measuring the levels of the ischemic stroke-related protein present in the sample. A difference in the level of an ischemic stroke-related protein in the sample, relative to the level of an ischemic stroke-related protein found an analogous sample from a subject who has not had an ischemic stroke, in particular examples indicates that the subject has suffered an ischemic stroke.
The solid support can be formed from an organic polymer. Suitable materials for the solid support include, but are not limited to: polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluroethylene, polyvinylidene difluroide, polyfluoroethylene-propylene, polyethylenevinyl alcohol, polymethylpentene, polycholorotrifluoroethylene, polysulfornes, hydroxylated biaxially oriented polypropylene, aminated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, etyleneacrylic acid, thylene methacrylic acid, and blends of copolymers thereof (see U.S. Pat. No. 5,985,567, herein incorporated by reference).
In general, suitable characteristics of the material that can be used to form the solid support surface include: being amenable to surface activation such that upon activation, the surface of the support is capable of covalently attaching a biomolecule such as an oligonucleotide thereto; amenability to “in situ” synthesis of biomolecules; being chemically inert such that at the areas on the support not occupied by the oligonucleotides are not amenable to non-specific binding, or when non-specific binding occurs, such materials can be readily removed from the surface without removing the oligonucleotides.
In one example, the solid support surface is polypropylene. Polypropylene is chemically inert and hydrophobic. Non-specific binding is generally avoidable, and detection sensitivity is improved. Polypropylene has good chemical resistance to a variety of organic acids (such as formic acid), organic agents (such as acetone or ethanol), bases (such as sodium hydroxide), salts (such as sodium chloride), oxidizing agents (such as peracetic acid), and mineral acids (such as hydrochloric acid). Polypropylene also provides a low fluorescence background, which minimizes background interference and increases the sensitivity of the signal of interest.
In another example, a surface activated organic polymer is used as the solid support surface. One example of a surface activated organic polymer is a polypropylene material aminated via radio frequency plasma discharge. Such materials are easily utilized for the attachment of nucleotide molecules. The amine groups on the activated organic polymers are reactive with nucleotide molecules such that the nucleotide molecules can be bound to the polymers. Other reactive groups can also be used, such as carboxylated, hydroxylated, thiolated, or active ester groups.
A wide variety of array formats can be employed in accordance with the present disclosure. One example includes a linear array of oligonucleotide bands, generally referred to in the art as a dipstick. Another suitable format includes a two-dimensional pattern of discrete cells (such as 4096 squares in a 64 by 64 array). As is appreciated by those skilled in the art, other array formats including, but not limited to slot (rectangular) and circular arrays are equally suitable for use (see U.S. Pat. No. 5,981,185, herein incorporated by reference). In one example, the array is formed on a polymer medium, which is a thread, membrane or film. An example of an organic polymer medium is a polypropylene sheet having a thickness on the order of about 1 mil. (0.001 inch) to about 20 mil., although the thickness of the film is not critical and can be varied over a fairly broad range. Particularly disclosed for preparation of arrays at this time are biaxially oriented polypropylene (BOPP) films; in addition to their durability, BOPP films exhibit a low background fluorescence.
The array formats of the present disclosure can be included in a variety of different types of formats. A “format” includes any format to which the solid support can be affixed, such as microtiter plates, test tubes, inorganic sheets, dipsticks, and the like. For example, when the solid support is a polypropylene thread, one or more polypropylene threads can be affixed to a plastic dipstick-type device; polypropylene membranes can be affixed to glass slides. The particular format is, in and of itself, unimportant. All that is necessary is that the solid support can be affixed thereto without affecting the functional behavior of the solid support or any biopolymer absorbed thereon, and that the format (such as the dipstick or slide) is stable to any materials into which the device is introduced (such as clinical samples and hybridization solutions).
The arrays of the present disclosure can be prepared by a variety of approaches. In one example, oligonucleotide or protein sequences are synthesized separately and then attached to a solid support (see U.S. Pat. No. 6,013,789, herein incorporated by reference). In another example, sequences are synthesized directly onto the support to provide the desired array (see U.S. Pat. No. 5,554,501, herein incorporated by reference). Suitable methods for covalently coupling oligonucleotides and proteins to a solid support and for directly synthesizing the oligonucleotides or proteins onto the support are known to those working in the field; a summary of suitable methods can be found in Matson et al., Anal. Biochem. 217:306-10, 1994. In one example, the oligonucleotides are synthesized onto the support using conventional chemical techniques for preparing oligonucleotides on solid supports (such as see PCT applications WO 85/01051 and WO 89/10977, or U.S. Pat. No. 5,554,501, herein incorporated by reference).
A suitable array can be produced using automated means to synthesize oligonucleotides in the cells of the array by laying down the precursors for the four bases in a predetermined pattern. Briefly, a multiple-channel automated chemical delivery system is employed to create oligonucleotide probe populations in parallel rows (corresponding in number to the number of channels in the delivery system) across the substrate. Following completion of oligonucleotide synthesis in a first direction, the substrate can then be rotated by 90° to permit synthesis to proceed within a second)(2° set of rows that are now perpendicular to the first set. This process creates a multiple-channel array whose intersection generates a plurality of discrete cells.
The oligonucleotides can be bound to the polypropylene support by either the 3′ end of the oligonucleotide or by the 5′ end of the oligonucleotide. In one example, the oligonucleotides are bound to the solid support by the 3′ end. However, one of skill in the art can determine whether the use of the 3′ end or the 5′ end of the oligonucleotide is suitable for bonding to the solid support. In general, the internal complementarity of an oligonucleotide probe in the region of the 3′ end and the 5′ end determines binding to the support.
In particular examples, the oligonucleotide probes on the array include one or more labels, that permit detection of oligonucleotide probe:target sequence hybridization complexes.
The nucleic acid molecules and proteins obtained from the subject (for example from a PBMC) can contain altered levels of one or more genes associated with ischemic stroke, such as those listed in Tables 2-5. Changes in expression can be detected to evaluate a stroke, or example to determine if the subject has had an ischemic stroke, to determine the severity of the stroke, to determine the likelihood of neurological recovery of a subject who has had an ischemic stroke, to determine the appropriate therapy for a subject who has had an ischemic stroke, or combinations thereof. The present disclosure is not limited to particular methods of detection. Any method of detecting a nucleic acid molecule or protein can be used, such as physical or functional assays. For example, the level of gene activation can be quantitated utilizing methods well known in the art and those disclosed herein, such as Northern-Blots, RNase protection assays, nucleic acid or antibody probe arrays, quantitative PCR (such as TaqMan assays), dot blot assays, in-situ hybridization, or combinations thereof. In addition, proteins can be quantitated using antibody probe arrays, quantitative spectroscopic methods (for example mass spectrometry, such as surface-enhanced laser desorption/ionization (SELDI)-based mass spectrometry), or combinations thereof.
Methods for labeling nucleic acid molecules and proteins so that they can be detected are well known. Examples of such labels include non-radiolabels and radiolabels. Non-radiolabels include, but are not limited to enzymes, chemiluminescent compounds, fluorophores, metal complexes, haptens, colorimetric agents, dyes, or combinations thereof. Radiolabels include, but are not limited to, 125I and 35S. Radioactive and fluorescent labeling methods, as well as other methods known in the art, are suitable for use with the present disclosure. In one example, the primers used to amplify the subject's nucleic acids are labeled (such as with biotin, a radiolabel, or a fluorophore). In another example, the amplified nucleic acid samples are end-labeled to form labeled amplified material. For example, amplified nucleic acid molecules can be labeled by including labeled nucleotides in the amplification reactions. In another example, nucleic acid molecules obtained from a subject are labeled, and applied to an array containing oligonucleotides. In a particular example, proteins obtained from a subject are labeled and subsequently analyzed, for example by applying them to an array.
The nucleic acid molecules obtained from the subject that are associated with ischemic stroke are applied to an ischemic stroke detection array under suitable hybridization conditions to form a hybridization complex. In particular examples, the nucleic acid molecules include a label. In one example, a pre-treatment solution of organic compounds, solutions that include organic compounds, or hot water, can be applied before hybridization (see U.S. Pat. No. 5,985,567, herein incorporated by reference).
Hybridization conditions for a given combination of array and target material can be optimized routinely in an empirical manner close to the Tm of the expected duplexes, thereby maximizing the discriminating power of the method. Identification of the location in the array, such as a cell, in which binding occurs, permits a rapid and accurate identification of sequences associated with ischemic stroke present in the amplified material (see below).
The hybridization conditions are selected to permit discrimination between matched and mismatched oligonucleotides. Hybridization conditions can be chosen to correspond to those known to be suitable in standard procedures for hybridization to filters and then optimized for use with the arrays of the disclosure. For example, conditions suitable for hybridization of one type of target would be adjusted for the use of other targets for the array. In particular, temperature is controlled to substantially eliminate formation of duplexes between sequences other than exactly complementary ischemic stroke-associated wild-type of mutant sequences. A variety of known hybridization solvents can be employed, the choice being dependent on considerations known to one of skill in the art (see U.S. Pat. No. 5,981,185, herein incorporated by reference).
Once the nucleic acid molecules associated with ischemic stroke from the subject have been hybridized with the oligonucleotides present in the ischemic stroke detection array, the presence of the hybridization complex can be analyzed, for example by detecting the complexes.
Detecting a hybridized complex in an array of oligonucleotide probes has been previously described (see U.S. Pat. No. 5,985,567, herein incorporated by reference). In one example, detection includes detecting one or more labels present on the oligonucleotides, the sequences obtained from the subject, or both. In particular examples, developing includes applying a buffer. In one example, the buffer is sodium saline citrate, sodium saline phosphate, tetramethylammonium chloride, sodium saline citrate in ethylenediaminetetra-acetic, sodium saline citrate in sodium dodecyl sulfate, sodium saline phosphate in ethylenediaminetetra-acetic, sodium saline phosphate in sodium dodecyl sulfate, tetramethylammonium chloride in ethylenediaminetetra-acetic, tetramethylammonium chloride in sodium dodecyl sulfate, or combinations thereof. However, other suitable buffer solutions can also be used.
Detection can further include treating the hybridized complex with a conjugating solution to effect conjugation or coupling of the hybridized complex with the detection label, and treating the conjugated, hybridized complex with a detection reagent. In one example, the conjugating solution includes streptavidin alkaline phosphatase, avidin alkaline phosphatase, or horseradish peroxidase. Specific, non-limiting examples of conjugating solutions include streptavidin alkaline phosphatase, avidin alkaline phosphatase, or horseradish peroxidase. The conjugated, hybridized complex can be treated with a detection reagent. In one example, the detection reagent includes enzyme-labeled fluorescence reagents or calorimetric reagents. In one specific non-limiting example, the detection reagent is enzyme-labeled fluorescence reagent (ELF) from Molecular Probes, Inc. (Eugene, Oreg.). The hybridized complex can then be placed on a detection device, such as an ultraviolet (UV) transilluminator (manufactured by UVP, Inc. of Upland, Calif.). The signal is developed and the increased signal intensity can be recorded with a recording device, such as a charge coupled device (CCD) camera (manufactured by Photometrics, Inc. of Tucson, Ariz.). In particular examples, these steps are not performed when fluorophores or radiolabels are used.
In particular examples, the method further includes quantification, for instance by determining the amount of hybridization.
The present disclosure provides for kits that can be used to evaluate a stroke, for example to determine if a subject has had an ischemic stroke, to determine the severity of the stroke, to determine the likelihood of neurological recovery of a subject who has had an ischemic stroke, to determine the appropriate therapy for a subject who has had an ischemic stroke, or combinations thereof. Such kits allow one to determine if a subject has a differential expression in ischemic stroke-related genes, such as any combination of four or more of those listed in Table 5, any combination of 150 or more of those listed in Table 3, or any combination of 500 or more of those listed in Table 2, for example any combination of at least one gene from each class of genes listed in Table 5 (such as at least 2 or at least 3 genes from each of the four classes of genes listed in Table 5).
The disclosed kits include a binding molecule, such as an oligonucleotide probe that selectively hybridizes to an ischemic stroke-related molecule that is the target of the kit. In particular examples, the oligonucleotides probes are attached to an array. In one example, the kit includes oligonucleotide probes or primers (or antibodies) that recognize any combination of at least four of the molecules in Table 5, such as at least 5, at least 10, at least 15, at least 20, or at least 22 of the ischemic stroke-related molecules listed in Table 5, such as any combination of at least 150 of the molecules in Table 3, such as at least 160, at least 170, at least 175, at least 180, at least 185, or at least 190 of the sequences listed in Table 3, such as any combination of at least 500 of the molecules in Table 2, such as at least 525, at least 550, at least 575, at least 600, at least 610, or at least 637 of the sequences listed in Table 2. In particular examples, the kit includes oligonucleotide probes or primers (or antibodies) that recognize at least one gene (or protein) from each class listed in Table 5, such as at least 2, at least 3, at least 5, or at least 10 genes from each class.
In one particular example, the kit includes oligonucleotide probes or primers (or antibodies) that recognize at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146. In one particular example, the kit includes oligonucleotide probes or primers (or antibodies) that recognize at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; or KIAA0146. In another particular example, the kit includes oligonucleotide probes or primers (or antibodies) that recognize CD163, for example in combination with oligonucleotide probes or primers (or antibodies) that recognize any combination of at least three ischemic stroke related molecules listed in Tables 2-5.
In a particular example, kits include antibodies capable of binding to ischemic stroke-related proteins. Such antibodies can be present on an array.
The kit can further include one or more of a buffer solution, a conjugating solution for developing the signal of interest, or a detection reagent for detecting the signal of interest, each in separate packaging, such as a container. In another example, the kit includes a plurality of ischemic stroke-related target nucleic acid sequences for hybridization with an ischemic stroke detection array to serve as positive control. The target nucleic acid sequences can include oligonucleotides such as DNA, RNA, and peptide-nucleic acid, or can include PCR fragments.
The present disclosure also provides methods of reducing brain injury in a subject determined to have suffered an ischemic stroke. For example, if using the assays described above a change in expression in at least 4 of the ischemic stroke-related molecules listed in Table 5 is detected in the subject, for example at least 22 of the ischemic stroke-related molecules listed in Table 5 is detected in the subject, a treatment is selected to avoid or reduce brain injury or to delay the onset of brain injury. In another example, if using the screening methods described above a change in expression in at least 500 of the ischemic stroke-related molecules listed in Table 2 is detected in the subject, a treatment is selected to avoid or reduce brain injury or to delay the onset of brain injury. The subject then can be treated in accordance with this selection, for example by administration of one or more anticoagulant agents. In some examples, the treatment selected is specific and tailored for the subject, based on the analysis of that subject's profile for one or more ischemic stroke-related molecules.
Based on the identification of multiple ischemic stroke-related molecules whose expression is altered following an ischemic stroke (such as those listed in Tables 2-5), the disclosure provides methods for identifying agents that can enhance, normalize, or reverse these effects. For example, the method permits identification of agents that normalize activity of an ischemic stroke-related molecule, such as a gene (or its corresponding protein) involved in vascular repair, response to hypoxia, response to altered cerebral microenvironment, or combinations thereof (see Table 5). Normalizing activity (such as the expression) of an ischemic stroke-related molecule can include decreasing activity of an ischemic stroke-related molecule whose activity is increased following an ischemic stroke, or increasing activity of an ischemic stroke-related molecule whose activity is decreased following an ischemic stroke. In another example, the method permits identification of agents that enhance the activity of an ischemic stroke-related molecule, such as an ischemic stroke-related molecule whose activity provides a protective effect to the subject following an ischemic stroke. For example, the method permits identification of agonists. In yet another example, the method permits identification of agents that decrease the activity of an ischemic stroke-related molecule, such as an ischemic stroke-related molecule whose activity results in one or more negative symptoms of ischemic stroke. For example, the method permits identification of antagonists.
In particular examples the identified agents can be used to treat a subject who has had an ischemic stroke, for example to alleviate or prevent one or more symptoms of an ischemic stroke, such as paralysis or memory loss.
The disclosed methods can be performed in vitro, for example by adding the test agent to cells in culture, or in vivo, for example by administering the test agent to a mammal (such as a human or a laboratory animal, for example a mouse, rat, dog, or rabbit). In particular examples, the method includes exposing the cell or mammal to conditions sufficient for mimicking an ischemic stroke. The one or more test agents are added to the cell culture or administered to the mammal under conditions sufficient to alter the activity of an ischemic stroke-related molecule, such as at least one of the molecules listed in Tables 2-5. Subsequently, the activity of the ischemic stroke-related molecule is determined, for example by measuring expression of one or more ischemic stroke-related molecules or by measuring an amount of biological activity of one or more ischemic stroke-related proteins. A change in the activity one or more ischemic stroke-related molecule indicates that the test agent alters the activity of an ischemic stroke-related molecule listed in Tables 2-5. In particular examples, the change in activity is determined by a comparison to a standard, such as an amount of activity present when no ischemic stroke has occurred, or an amount of activity present when an ischemic stroke has occurred, or to a control.
Any suitable compound or composition can be used as a test agent, such as organic or inorganic chemicals, including aromatics, fatty acids, and carbohydrates; peptides, including monoclonal antibodies, polyclonal antibodies, and other specific binding agents; phosphopeptides; or nucleic acid molecules. In a particular example, the test agent includes a random peptide library (for example see Lam et al., Nature 354:82-4, 1991), random or partially degenerate, directed phosphopeptide libraries (for example see Songyang et al., Cell 72:767-78, 1993). A test agent can also include a complex mixture or “cocktail” of molecules.
Therapeutic agents identified with the disclosed approaches can be used as lead compounds to identify other agents having even greater desired activity. In addition, chemical analogs of identified chemical entities, or variants, fragments, or fusions of peptide test agents, can be tested for their ability to alter activity of an ischemic stroke-related molecule using the disclosed assays. Candidate agents can be tested for safety in animals and then used for clinical trials in animals or humans.
In one example, the method is an in vitro assay. For example, cells, such as cells that can provide a model of what happens in vivo following an ischemic stroke, are cultured under conditions sufficient for mimicking an ischemic stroke, such as hypoxia, hypoglycemia, or combinations thereof. Simultaneously or at a time thereafter, one or more test agents are incubated with the cells under conditions sufficient for the test agent to have the desired effect on the cell, for example to alter (such as normalize) the activity of a ischemic stroke-related molecule. In particular examples, the test agent has the desired effect on more than one ischemic stroke-related molecule.
Examples of cells that can be used include, but are not limited to: PBMCs, endothelial cells, neuronal cells, or combinations thereof. Methods of isolating PBMCs from a subject are disclosed herein. Neuronal cells and endothelial cells can also be obtained from a subject, such as a mammal, and grown as a primary culture using standard methods. For example, endothelial cells can be obtained from umbilical cord tissue (for example see Ulrich-Merzenich et al., In Vitro Cellular & Developmental Biology-Animal, 38: 265-72, 2002); coronary arteries (Dame et al., In Vitro Cellular & Developmental Biology-Animal, 39:402-6, 2003); or lung tissue (for example see Dong et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 17:1599-604, 1997). For example, neuronal cells can be obtained from mammalian brain tissue (for example see Shevtsova et al., Exp. Physiol. 90:53-9, 2005 and Buse et al., Brain Res. 283:221-34, 1983). In one example, established neuronal or endothelial tissue culture cell lines are used, such as those available from American Type Culture Collection (ATCC) and other commercial sources. For example, rat PC12 pheochromocytoma neurosecretory cell line (ATCC No. CRL-1721), human neuronal HCN-2 cells (ATCC No. CRL-10742), the rat neuronal RSC96 cell line (CRL-2765), human HAAE-2 endothelial cells (ATCC No. CRL-2473), human HPAE-26 endothelial cells (ATCC No. CRL-2598), human aortic endothelial cells (Clonetics), and bovine FBHE endothelial cells (ATCC No. CRL-1395) are particular examples of cell lines that can be used. However one skilled in the art will appreciate that other cell lines can be used.
Methods of providing conditions sufficient for mimicking an ischemic stroke in vitro are known in the art. For example, cells can be exposed to hypoxic conditions (low oxygen) by culturing the cells in an atmosphere controlled-culture chamber (for example a chamber from Bellco Glass [Vineland, N.J.]; a modular hypoxia chamber [Billups-Rothenberg]; an Espec [Grand Rapids, Mich.]; or BIO-LABO [Juji Field, Tokyo, Japan]. A particular example of hypoxic conditions is a chamber containing a gas mixture of 94% N2, 5% CO2, and 1% O2. Cells are generally grown at 37° C. The amount of time the cells are exposed to hypoxic conditions can vary. In particular examples, cells are exposed to hypoxic conditions for at least 10 minutes, such as at least 30 minutes, at least 1 hour, at least 6 hours, at least 12 hours, or even at least 24 hours. In one example, hypoxic conditions are used to identify free radical scavenger agents.
Another method that can be used to mimic an ischemic stroke is to expose the cells to hypoglycemic conditions (low glucose). Hypoglycemia (such as <30 mg glucose/dl) can result from ischemia (for example using the conditions described above), or can be induced by culturing cells in growth medium that does not contain added glucose. Cells are generally grown at 37° C. The amount of time the cells are exposed to hypoglycemic conditions (deprived of glucose) can vary. In particular examples, cells are exposed to hypoglycemic conditions for at least 1 hour, such as at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, or even at least 72 hours. In some examples, the hypoglycemic conditions are combined with hypoxic conditions.
One or more test agents are incubated with the cells under conditions sufficient for the test agent to have the desired effect on the cell. The agent can be added at a time subsequent to mimicking an ischemic stroke, or at substantially the same time as mimicking an ischemic stroke. In one example, the agent is added at least 30 minutes after mimicking an ischemic stroke, such as at least 1 hour, at least 2 hours, at least 6 hours, at least 24 hours, at least 72 hours, at least 7 days, at least 14 days, at least 30, days, at least 60 days or even at least 90 days after mimicking an ischemic stroke.
In another example, the method is an in vivo assay. For example, agents identified as candidates in the in vitro assay can be tested in vivo for their ability to alter (such as normalize) the activity of a ischemic stroke-related molecule (such as one or more of those listed in Tables 2-5). In particular examples, the mammal has had an ischemic stroke or has been exposed to conditions that induce an ischemic stroke. Simultaneously or at a time thereafter, one or more test agents are administered to the subject under conditions sufficient for the test agent to have the desired effect on the subject, for example to alter (such as normalize) the activity of an ischemic stroke-related molecule or a pattern of ischemic stroke-related molecules. In particular examples, the test agent has the desired effect on more than one ischemic stroke-related molecule.
Methods of providing conditions sufficient for inducing an ischemic stroke in vivo are known in the art. For example, ischemic stroke can be induced in a mammal by occlusion of the middle cerebral artery (MCA) under anesthesia. Ischemic stroke can also be induced in a mammal (such as a rat), for example by three-vessel (bilateral vertebral and unilateral common carotid artery) occlusion (3-VO) to induce unilaterally accentuated brain hypoperfusion under anesthesia (for example using the method described in Busch et al., J. Cereb. Blood Flow Metab. 23:621-8, 2003) or by the four-vessel occlusion (4-VO) method to induce transient forebrain ischemia. In another example, the subject is exposed hypoxic conditions. For example, a mammal can be exposed to sublethal hypoxia conditions, such as 11% oxygen for 2 hours. In another example, the right carotid artery is ligated and mammal exposed to 8% oxygen for 2 hours.
One or more test agents are administered to the subject under conditions sufficient for the test agent to have the desired effect on the subject. Any appropriate method of administration can be used, such as intravenous, intramuscular, or transdermal. The agent can be administered at a time subsequent to the ischemic stroke, or at substantially the same time as the ischemic stroke. In one example, the agent is added at least 30 minutes after the ischemic stroke, such as at least 1 hour, at least 2 hours, at least 6 hours, at least 24 hours, at least 72 hours, at least 7 days, at least 14 days, at least 30 days, at least 60 days or even at least 90 days after the ischemic stroke.
The effect on the one or more test agents on the activity of one or more ischemic stroke-related molecules can be determined using methods known in the art. For example, the effect on expression of one or more ischemic stroke-related genes can be determined using the arrays and methods disclosed herein. For example, RNA can be isolated from the cultured cells exposed to the test agent, or from cells obtained from a subject (such as PBMCs) administered the test agent. The isolated RNA can be labeled and exposed to an array containing one or more nucleic acid molecules (such as a primer or probe) that can specifically hybridize to one or more pre-selected ischemic stroke-related genes, such at least 1, at least 2, or at least 3 of those listed in Tables 2-5, or to a pre-selected pattern of such genes that is associated with ischemic stroke. In a particular example, the one or more pre-selected ischemic stroke-related genes include at least one gene involved in vascular repair, at least one response to hypoxia gene, at least one response to altered cerebral microenvironment gene, or combinations thereof (for example see Table 5). In another example, proteins are isolated from the cultured cells exposed to the test agent, or from cells obtained from a subject (such as PBMCs) administered the test agent. The isolated proteins can be analyzed to determine amounts of expression or biological activity of one or more ischemic stroke-related proteins, such at least 1, at least 2, or at least 3 of those listed in Tables 2-5, or a pattern of upregulation or downregulation of pre-identified or pre-selected proteins. In a particular example, the one or more pre-selected ischemic stroke-related proteins include at least one protein involved in vascular repair, at least one response to hypoxia protein, at least one response to altered cerebral microenvironment protein, or combinations thereof (for example see Table 5). In a particular example, mass spectrometry is used to analyze the proteins.
In particular examples, differential expression of an ischemic stroke-related molecule is compared to a standard or a control. One example of a control includes the amount of activity of an ischemic stroke-related molecule present or expected in a subject who has not had an ischemic stroke, wherein an increase or decrease in activity in a test sample of an ischemic stroke-related molecule (such as those listed in Tables 2-5) compared to the control indicates that the test agent alters the activity of at least one ischemic stroke-related molecule. Another example of a control includes the amount of activity of an ischemic stroke-related molecule present or expected in a subject who has had an ischemic stroke, wherein an increase or decrease in activity in a test sample (such as gene expression, amount of protein, or biological activity of a protein) of an ischemic stroke-related molecule (such as those listed in Tables 2-5) compared to the control indicates that the test agent alters the activity of at least one ischemic stroke-related molecule. Detecting differential expression can include measuring a change in gene expression, measuring an amount of protein, or determining an amount of the biological activity of a protein present.
In particular examples, test agents that altered the activity of an ischemic stroke-related molecule are selected.
The disclosure is further illustrated by the following non-limiting Examples.
This example describes methods used to obtain RNA from control subjects (subjects who had not previously had a stroke) and subjects who suffered an ischemic stroke within the previous 72 hours.
A cohort of elderly volunteers was obtained and their stroke risk factors recorded, including a history of hypertension, smoking, diabetes mellitus and heart disease. Approximately 30 milliliters of blood was drawn into four yellow top ACD A tubes (ACD Acid Citrate Dextrose A, 22.0 g/L Trisodium Citrate, 8.0 g/L Citric Acid, 24.5 g/L Dextrose, BD Franklin Lakes, N.J.) by aseptic antecubital fossa venipuncture. PBMC isolation was completed within two hours.
Acute stroke patients admitted to the National Institutes of Health Stroke Program at Suburban Hospital in Bethesda, Md. underwent aseptic antebrachial venipuncture followed by withdrawal of 30 ml of blood as described above. Blood samples were drawn within 72 hours of stroke onset. The blood samples were processed for RNA within two hours of collection.
Table 1 lists the demographic features of the patients and controls in the index cohort (n=38) and the patients and controls in the validation (test) cohort (n=19). The two index groups are reasonably comparable in terms of age sex and pre-morbid risk factors consistent with a community based stroke population.
Acute stroke was confirmed by magnetic resonance imaging studies including diffusion weighted imaging (DWI) and perfusion imaging. Stroke risk factors were recorded on each patient and volunteer according to the Framingham risk profile (see Wolf et al., Stroke 22:312-8, 1991). Stroke severity was determined by serial neurological examination and by the National Institutes of Health Stroke Scale (NIHSS) score (see Brott et al., Stroke 20:871-5, 1989).
RNA was isolated from PBMCs as follows. Total RNA (5-15 μg) was extracted from PBMCs separated from whole blood using a Density Gradient tube (Uni-Sep, Novamed, Jerusalem, Israel) as follows: 20-30 ml ACD anti-coagulated blood was diluted with an equal volume of phosphate buffered saline (PBS) and added to the density gradient tube, followed by centrifugation at 1000 g for 30 minutes. After centrifugation, the PBMC layer was removed.
RNA was extracted using RNeasy Mini Kit (Qiagen Cat. #75162, Valencia, Calif.), as per the manufacturer's protocol. Briefly, harvested PBMCs are diluted 1:1 with PBS and centrifuged for 10 minutes at 4000 rpm. The resulting supernatant was discarded and the pellet resuspended in 600 μl RLT buffer (1 ml buffer+10 μl 2-β-mercaptoethanol). The sample was homogenized by passing the lysate 5-10 times through 20-G (French) needle fitted to a syringe. Cells were resuspended in 600 μl of DEPC-H2O diluted in 70% EtOH and was loaded onto an RNeasy mini spin column fitted with a 2-ml collection tube. The sample was twice centrifuged at 14,000 rpm for 15 seconds. The RNeasy column was transferred to a new 2 ml collection tube and 500 μl of RPE buffer added followed by centrifugation at 14,000 rpm for 15 seconds. RPE buffer (5000 was added and the sample centrifuged at 10,000 rpm for 2 minutes. The RNeasy column was then transferred into a new 1.5 ml collection tube and RNA free water (30 μl) directly added to the RNase membrane followed by further centrifugation at 10,000 rpm for 1 minute. This was repeated and the extracted RNA stored at −80° C.
This example describes methods used to label the RNA obtained in Example 1. However, one skilled in the art will appreciate that other labels and methods can be used.
RNA obtained from PBMCs was biotin-labeled and cleaned according to Affymetrix guidelines for Human Genome 133A arrays. Briefly, the Enzo BioArray HighYield RNA Transcript Labeling Kit3 (Affymetrix, P/N 900182) was used for generating labeled cRNA target. Template cDNA and the other reaction components were added to RNase-free microfuge tubes. To avoid precipitation of DTT, reactions were at room temperature while additions were made. After adding all reagents, the tube was incubated are a 37° C. for 4 to 5 hours, gently mixing the contents of the tube every 30-45 minutes during the incubation.
To ensure the quality of the initial isolated total RNA, DNase was used to remove contaminant DNA from the sample. In addition, Northern blot followed by optical density analysis was used to determine the concentration of the RNA band.
If the total RNA concentration was >5 μg, the RNA was used for subsequent gene chip hybridization as per the manufacturer's protocol.
Coded mRNA samples were analyzed using the Affymetrix GeneChipR Human Genome U133A chips that include 22,283 gene probes (around 19,000 genes) of the best characterized human genes. Microarrays were scanned (Axon scanner, Axon Instruments Inc, CA), and images were analyzed using GenePix image analysis software (Axon Instruments Inc, CA) allowing for gene spot fluorescent quantification following subtraction of the surrounding background fluorescent signal within the
Affymetrix MASS gene chip analysis suite with production of .CEL, and .DAT output files. The .CDF file or annotation file for the Affymetrix HU133A chip and the .CEL files, containing the scanned gene expression information, were the only data files used in all subsequent analyses.
For the data analysis, .CEL files of 19 patients and 19 controls were used following exclusion from analysis of one chip in each of the index patient and control groups due to unsatisfactory hybridization (see Irizarry et al., The Analysis of Gene Expression Data. New York: Springer, 2003). The analysis was completed using the Bioconductor applications of the R programming language and implemented on a 64-bit operating system (SGI Octane 14000 MIPS 600 MHz CPU running Irix 6.5.15) due to the large dataset for analysis (Moore et al., 32 bit architecture—a severe bio-informatics limitation. NHLBI Symposium From Genome to Disease. 2003, Bethesda, Md.: 64). Sample RNA degradation during processing was tightly distributed and uniform across all chips.
Quantile normalization was performed simultaneously on the .CEL dataset (stroke patients, n=19, controls, n=19).
Following normalization, expression levels for each gene were calculated using the perfect match array probes and a robust median polish technique after background correction and log2 transformation (Irizarry et al., The Analysis of Gene Expression Data. New York: Springer, 2003). The resulting expression set was compared in a univariate manner between the stroke patients and control group using parametric testing (t-test). The uncorrected p-value were assigned a cutoff threshold value of significance of <0.05. Subsequent multiple comparison correction was performed using Bonferroni and false discovery techniques (Benjamini and Yekutieli, The Annals of Statistics 29:1165-88, 2001). The effects of various multiple comparison correction techniques are shown in
The uncorrected significant gene expression set was further analyzed using permutation analysis of Westfall and Young (Resampling-based multiple testing: Examples and methods for p-value adjustment. New York: John Wiley & Sons, 1993). Hierarchical cluster analysis was performed on the gene subset found to be significantly different between stroke patients and controls using the method of Eisen et al. where each gene was pair-wise correlated by calculation of a distance matrix using a Euclidean metric (Proc. Natl. Acad. Sci. 95:14863-8, 1998). The distance matrix then formed the basis for hierarchical clustering. Gene annotation and ontology were determined using the Affymetrix on-line NetAffix suite together with subsequent literature searches, allowing categorization of a gene listing into molecular function, cellular function and biological function.
Using the PAM algorithm (Prediction Analysis for Microarrays) the ability of the index set to separate prospectively obtained samples from ten stroke patients and ten controls was examined (Tibshirani et al., Proc. Natl. Acad. Sci. 90:6567-72, 2002). The arrays of 9 patients and 10 controls were used. In one stroke case, the hybridization was not of sufficient quality to be included.
Without multiple comparison correction, 5060 genes were significantly different in the dataset. The Benjamini and Yekutieli correction resulted in 771 significant gene probes (Table 2), which represent 637 genes. This approach seeks to limit the false discovery rate (the proportion of non-differentiated genes among all those genes declared significantly different) to 5%. As shown in Table 2, several genes were upregulated (positive T-statistic, such as a value that is at least 3.77) or downregulated (negative t-statistic, such as a value that is less than −3.76, such as less than −3.77) following an ischemic stroke. In addition, several genes not previously associated with ischemic stroke, such as CD163; hypothetical protein FLJ22662 Laminin A motif; bone marrow stromal cell antigen 1/CD157; Fc fragment of IgG, high affinity Ia, receptor for (CD64); baculoviral IAP repeat-containing protein 1; and KIAA0146, were identified.
Homo sapiens transcribed sequence with strong
Homo sapiens similar to neutrophil cytosolic factor
Homo sapiens transcribed sequence with strong
Homo sapiens transcribed sequence with strong
Homo sapiens mRNA; cDNA DKFZp586I1823
Homo sapiens cDNA FLJ44314 fis, clone
Homo sapiens transcribed sequence with strong
Homo sapiens T cell receptor beta chain BV20S1
Homo sapiens T cell receptor beta chain BV20S1
Homo sapiens cDNA FLJ11958 fis, clone
Following Bonferroni correction, 231 gene probes, corresponding to 190 genes, were found to be significant (Table 3). Clear separation of the stroke and control gene expression levels were observed. As shown in Table 3, several genes were upregulated (positive T-statistic, such as a value that is at least 4.73) or downregulated (negative t-statistic, such as a value that is less than −4.73) following an ischemic stroke.
Homo sapiens transcribed sequence with strong
Homo sapiens cDNA FLJ11958 fis, clone
After multiple comparison correction (MCC) using the Westfall and Young permutation approach, 91 gene probes, corresponding to 82 genes were found to be significantly different (Table 4). As shown in Table 4, several genes were upregulated (positive T-statistic, such as a value that is at least 5.3) or downregulated (negative t-statistic, such as a value that is less than −5.4) following an ischemic stroke.
Homo sapiens transcribed sequence with
In contrast to the Benjamini and Yekutieli approach, the Westfall and Young approach limits the probability of making even one false positive declaration at 5%. There was a predominant up-regulation pattern with 77/82 genes up-regulated and 5 down-regulated (Table 4).
After PAM correction, 28 gene probes, corresponding to 22 genes were found to be significantly different (Table 5). As shown in Table 5, several genes were upregulated following an ischemic stroke.
Table 6 provides a summary of the number of ischemic-stroke related genes found using different correction methods.
This example describes the four classes of genes whose expression was increased following ischemic stroke, based on the results obtained in Example 3.
A number of broad classes of gene expression were found (representative examples are shown in Table 5 above). The first were genes that indicated differentiation of monocytes into macrophages and lymphocyte activation (for example, CD14, toll-like receptor 2 and FcR2a). Concomitantly, a number of genes for cell cycle arrest were up-regulated. Some other up-regulated genes were for cytoskeletal proteins (for example, alpha-catenin and galectin 3) involved in anchoring of white blood cells to tissue.
The second main grouping was related to hypoxia, many being inducible by hypoxia inducible factor-1 (for example, adrenomedullin, FcR2a and CD14). There may be a common promoter region for hypoxia inducible factor-1.
A third class of genes is related to vascular repair. For example, up-regulation of ectonucleoside triphosphate diphosphohydrolase 1 results in decreased platelet interaction and aggregation.
The fourth broad class of genes is related to a specific PBMC response to the altered cerebral microenvironment.
Surprisingly, no specific steroid stress-related genes were identified.
In summary, the gene classes demonstrate both specific and non-specific gene expression in PBMCs during acute ischemic stroke. The finding of genes induced by hypoxic stress, vascular repair genes and neuronal specific genes demonstrates a specific response to ischemic stroke.
This example describes methods used to analyze PBMCs isolated from 26 subjects at three time-points following ischemic stroke, to demonstrate that there is a correlation between recovery and alterations in gene expression.
Expression of the 22 genes listed in Table 5 was determined using the methods described in the above examples in a second and independent series of 26 patients studied two years after the initial series. These patients had blood samples drawn at day 1 (within 24 hours of onset of symptoms), day 7-14 and day 90 post stroke (26 subjects had blood draws at day 1, 25 subjects had a blood draw at day 7-14 and 21 subjects had a blood draw at day 90 [some patients were deceased by this time]). At day 1, detecting differential gene expression in the 22 genes accurately classified 81% of subjects (21/26) as having had an ischemic stroke. The 5 subjects classified as control (that is, subjects classified as not having had an ischemic stroke) using the method tended to be younger or to have mild stroke severity scores. These results confirm the diagnostic accuracy of the PAM list (Table 5) for acute stroke diagnosis (shown in Table 9), as this was the second independent series of subjects on which these results have been confirmed.
At days 7-14, detecting differential gene expression in the 22 genes accurately classified 64% of subjects (16/25) as having had an ischemic stroke. At day 90, detecting differential gene expression in the 22 genes accurately classified 62% of subjects (13/21) as having had an ischemic stroke. Without wishing to be bound to a particular theory, it is proposed that the persistent gene changes of ischemic stroke at the day 7-14 and day 90 time points reflects ongoing inflammatory or other processes related to the stroke or a lack of recovery of these processes. Those who remained classified as a stroke at these time points were those with the more severe strokes and worse outcomes (see below).
The recovery of the subjects was compared to their classification determined using the 22 genes listed in Table 5. An excellent recovery was defined as a Barthel score of 100 at three months post stroke (for example see Mahoney et al., Md. State Med. J. 14:61-5, 1965). The Barthel score is a measure of 10 activities of daily living such as getting dressed, walking, going to the toilet. The score ranges from 100 (fully independent) to 0 (totally dependent and incapacitated or deceased).
In terms of excellent stroke recovery, all 9/9 (100%) patients who were classified as a control at their last measurement (whether classified as a stroke or a control at the first time point) had excellent recovery. In contrast only 8/17 (47%) patients who remained classified as a stroke at their last follow-up measurement (at day 7-14 in 3 patients who died and day 90 in the remaining) had excellent stroke recovery (p=0.008). This indicates persistence of the stroke state is related to changes in gene expression.
Therefore, it appears that the reason that some of the subjects were indicated to not have had an ischemic stroke is that they recovered by day 90. Therefore, the disclosed ischemic stroke related molecules, such as those listed in Tables 2-5, for example those listed in Table 5 can be used to be determine the prognosis of a subject who has had an ischemic stroke.
In view of these results, disclosed are methods of stratifying the seriousness of a stroke, and assessing the likely neurological recovery of the subject. For example, stratification or assessing the likely neurological recovery of the subject can be performed as early as one day (or within 24 hours) after the ischemic stroke, 7-14 days after the ischemic stroke, or 90 days after the ischemic stroke. In particular examples, the method includes detecting differential expression in at least four ischemic stroke-related molecules, such as at least the 22 genes (or corresponding proteins) listed in Table 5. Detection of increased expression of at least four ischemic stroke-related molecules, such as at least the 22 genes (or corresponding proteins) listed in Table 5, indicates that the stroke was severe and the subject has a lower probability of neurological recovery (for example as compared to an amount of expected neurological recovery in a subject who did not have increased expression of the 22 genes/proteins listed in Table 5). In particular examples, the increased expression is determined by calculating a t-statistic value, wherein a t-statistic value of at least 3, at least 5.3, or at least 6 indicates that expression is increased.
In particular examples, the assay results can predict a Barthel score of at least 45, for example at least 50, 90 or 100, as an indication of neurological recovery.
This example describes the temporal relationship of the disclosed methods to the stroke or suspected stroke. The assay can be performed following the onset of signs and symptoms associated with ischemic stroke. Particular examples of signs and symptoms associated with ischemic stroke include but are not limited to: headache, sensory loss (such as numbness, particularly confined to one side of the body or face), paralysis (such as hemiparesis), pupillary changes, blindness (including bilateral blindness), ataxia, memory impairment, dysarthria, somnolence, and other effects on the central nervous system recognized by those of skill in the art.
A sample can be obtained from the subject (such as a PBMC sample) and analyzed using the disclosed methods, for example, within 1 hour, within 6 hours, within 12 hours, or even within 24 hours of having signs or symptoms associated with ischemic stroke, In another example, a sample is obtained at least 7 days later following the onset of signs and symptoms associated with ischemic stroke, such as within 7-14 days of having signs or symptoms associated with ischemic stroke, or within 90 days.
In particular examples, the assay can be performed after a sufficient period of time for the differential regulation of the genes (or proteins) to occur, for example at least 24 hours after onset of the symptom or constellation of symptoms that have indicated a potential cerebral ischemic event. In other examples it occurs prior to performing any imaging tests are performed to find anatomic evidence of ischemic stroke. Moreover, it is often difficult for imaging modalities (such as CT and MRI) to detect acute ischemic strokes, at least until brain changes (such as edema) have taken place in response to the ischemia. Hence the assay described herein in particular examples is able to detect the ischemic stroke even before definitive brain imaging evidence of the stroke is known.
Since the results of this assay are also highly reliable predictors of the ischemic nature of the stroke, the results of the assay can also be used (for example in combination with other clinical evidence and brain scans) to determine whether thrombolytic therapy designed to lyse a neurovascular occlusion such as a thrombus (for example by using tissue plasminogen activator or streptokinase) should be administered to the subject. In certain example, thrombolytic therapy is given to the subject once the results of the differential gene assay are known if the assay provides an indication that the stroke is ischemic in nature.
Moreover, the neurological sequelae of an ischemic event in the central nervous system can have consequences that range from the insignificant to the devastating, and the disclosed assay permits early and accurate stratification of risk of long-lasting neurological impairment. For example, a test performed as early as within the first 24 hours of onset of signs and symptoms of a stroke, and even as late as 7-14 days or even as late as 90 days or more after the event can provide clinical data that is highly predictive of the eventual care needs of the subject.
The disclosed assay is also able to identify subjects who have had an ischemic stroke in the past, for example more than 2 weeks ago, or even more than 90 days ago. The identification of such subjects helps evaluate other clinical data (such as neurological impairment or brain imaging information) to determine whether an ischemic stroke has occurred. Subjects identified or evaluated in this manner can then be provided with appropriate treatments, such as anti-platelet agents (for example aspirin) that would be appropriate for a subject identified as having had an ischemic stroke but not as appropriate for subject who have had a hemorrhagic stroke. It is helpful to be able to classify subject as having had an ischemic stroke, because the treatments for ischemic stroke are often distinct from the treatments for hemorrhagic stroke. In fact, treating a hemorrhagic stroke with a therapy designed for an ischemic stroke (such as a thrombolytic agent) can have devastating clinical consequences. Hence using the results of the disclosed assay to help distinguish ischemic from hemorrhagic stroke offers substantial clinical benefit, and allows subjects to be selected for treatments appropriate to ischemic stroke but not hemorrhagic stroke.
This example describes the use of quantitative real time polymerase chain reaction to confirm results obtained using the microarrays.
Quantitative real time polymerase chain reaction of gene expression levels were performed using RNA samples from 10 patients and 9 controls. Nine genes were selected for analysis on the basis of their significantly high expression in the index set. One further gene, not up-regulated in the permutation dataset was selected as a negative control. Primers were obtained from the published literature and ordered from Invitrogen (Carlsbad, Calif.) as listed in Table 7.
Real time PCR was performed with an Opticon 2 (MJ research). Real time PCR results between patients and controls were compared using non-parametric statistics (Mann Whitney U tests).
As shown in Table 8, expression values derived from the microarrays correlated with RT-PCR for 9 up-regulated genes. Using RT-PCR, higher values for 8/9 genes in the up-regulated list were found, with a significant difference in 7/9 genes between 10 patients and 9 controls. A negative control was also included (gene not up-regulated in the permutation dataset) with no significant difference observed between patients who suffered a stroke and controls.
Using data from 9 patients and 10 controls and the PAM, stroke was prospectively classified with a sensitivity of 78% and a specificity of 80% (Table 9).
In summary, a distinct genomic profile of acute ischemic stroke in the peripheral blood mononuclear cells was identified. In addition, four broad classes of ischemic stroke related genes were identified that are upregulated following an ischemic stroke: white blood cell activation and differentiation genes, genes associated with hypoxia, vascular repair genes and genes associated with an altered cerebral microenvironment, including neuronal apoptosis inhibitory protein.
This example describes particular arrays that can be used to evaluate a stroke, for example to diagnose an ischemic stroke.
In one example, the array includes probes (such as an oligonucleotide or antibody) that can recognize at least one gene (or protein) that is upregulated following an ischemic stroke, such as one or more of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, or any 1, 2, 3, 4, 5, or 6 of these. For example, the array can include a probe (such as an oligonucleotide or antibody) recognizes CD163. In yet another example, the array includes probes (such as an oligonucleotide or antibody) that can recognize at least one gene (or protein) that is downregulated following an ischemic stroke, such as one or more of intercellular adhesion molecule 2; protein kinase D2; GATA binding protein 3; hypothetical protein FLJ20257; or protein kinase C, theta. In a particular example, the array includes probes (such as an oligonucleotide or antibody) that can recognize at least one gene (or protein) that is upregulated following an ischemic stroke (such as at least one of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146) and at least one gene (or protein) that is downregulated following an ischemic stroke (such as one or more of intercellular adhesion molecule 2; protein kinase D2; GATA binding protein 3; hypothetical protein FLJ20257; or protein kinase C, theta).
Other examplary probes that can be used are listed in Tables 2-5 and are identified by their Affymetrix identification number. The disclosed oligonucleotide probes can further include one or more detectable labels, to permit detection of hybridization signals between the probe and a target sequence.
In one example, the array includes probes (such as an oligonucleotide or antibody) that recognize any combination of at least four different genes (or proteins) listed in Tables 2-5. In particular examples, the array includes probes recognize all 22 genes (or proteins) listed in Table 5. The accuracy of the PAM list (Table 5) to diagnose ischemic acute stroke has been confirmed in two independent series of subjects. The ability of the PAM list (Table 5) to provide an indication of the severity of the stroke and to determine the likelihood of neurological recovery has also been demonstrated. In some examples, the array includes oligonucleotides, proteins, or antibodies that recognize any combination of at least one gene from each of the four classes listed in Table 5 (such as at least 2 or at least 3 genes from each class).
In another example, the array includes probes (such as an oligonucleotide or antibody) that recognize any combination of at least 150 different genes listed in Table 3 or all 190 genes listed in Table 3. In yet another example, the array includes probes that recognize at least 500 different genes listed in Table 2. In particular examples, the probes recognize all 637 genes listed in Table 2.
Compilation of “loss” and “gain” of hybridization signals will reveal the genetic status of the individual with respect to the ischemic stroke-associated genes listed in Tables 2-5.
This example describes quantitative spectroscopic approaches methods, such as SELDI, that can be used to detect differential protein expression of ischemic stroke related proteins.
In one example, surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry is used to detect changes in differential protein expression, for example by using the ProteinChip™ (Ciphergen Biosystems, Palo Alto, Calif.). Such methods are well known in the art (for example see U.S. Pat. No. 5,719,060; U.S. Pat. No. 6,897,072; and U.S. Pat. No. 6,881,586, all herein incorporated by reference). SELDI is a solid phase method for desorption in which the analyte is presented to the energy stream on a surface that enhances analyte capture or desorption.
Briefly, one version of SELDI uses a chromatographic surface with a chemistry that selectively captures analytes of interest, such as ischemic stroke related proteins. Chromatographic surfaces can be composed of hydrophobic, hydrophilic, ion exchange, immobilized metal, or other chemistries. For example, the surface chemistry can include binding functionalities based on oxygen-dependent, carbon-dependent, sulfur-dependent, and/or nitrogen-dependent means of covalent or noncovalent immobilization of analytes. The activated surfaces are used to covalently immobilize specific “bait” molecules such as antibodies, receptors, or oligonucleotides often used for biomolecular interaction studies such as protein-protein and protein-DNA interactions.
The surface chemistry allows the bound analytes to be retained and unbound materials to be washed away. Subsequently, analytes bound to the surface (such as ischemic stroke related proteins) can be desorbed and analyzed by any of several means, for example using mass spectrometry. When the analyte is ionized in the process of desorption, such as in laser desorption/ionization mass spectrometry, the detector can be an ion detector. Mass spectrometers generally include means for determining the time-of-flight of desorbed ions. This information is converted to mass. However, one need not determine the mass of desorbed ions to resolve and detect them: the fact that ionized analytes strike the detector at different times provides detection and resolution of them. Alternatively, the analyte can be detectably labeled (for example with a fluorophore or radioactive isotope). In these cases, the detector can be a fluorescence or radioactivity detector. A plurality of detection means can be implemented in series to fully interrogate the analyte components and function associated with retained molecules at each location in the array.
Therefore, in a particular example, the chromatographic surface includes antibodies that recognize ischemic stroke related proteins. In one example, antibodies are immobilized onto the surface using a bacterial Fc binding support. The chromatographic surface is incubated with a sample from the subject, such as a sample that includes PMBC proteins (such as a PBMC lysate). The antigens present in the sample can recognize the antibodies on the chromatographic surface. The unbound proteins and mass spectrometric interfering compounds are washed away and the proteins that are retained on the chromatographic surface are analyzed and detected by SELDI-TOF. The MS profile from the sample can be then compared using differential protein expression mapping, whereby relative expression levels of proteins at specific molecular weights are compared by a variety of statistical techniques and bioinformatic software systems.
The ischemic stroke-related nucleic acid molecules provided herein (such as those disclosed in Tables 2-5) can be used in evaluating a stroke, for example for determining whether a subject has had an ischemic stroke, determining the severity or likely neurological recovery of a subject who has had an ischemic stroke, and determining a treatment regimen for a subject who has had an ischemic stroke. For such procedures, a biological sample of the subject is assayed for an increase or decrease in expression of ischemic stroke-related nucleic acid molecules, such as those listed in Tables 2-5. Suitable biological samples include samples containing genomic DNA or RNA (including mRNA) obtained from cells of a subject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. In a particular example, the sample includes PBMCs (or components thereof, such as nucleic acids or proteins isolated from PBMCs).
The detection in the biological sample of increased or decreased expression in four or more ischemic stroke-related nucleic acid molecules, such any combination of four or more molecules listed in Table 5, 150 or more molecules listed in Table 3, or 500 or more molecules listed in Table 2, can be achieved by methods known in the art. In some examples, expression is determined for any combination of at least one gene from each class listed in Table 5 (such as at least 2 or at least 3 genes from each class). In some examples, expression is determined for at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146.
Increased or decreased expression of an ischemic stroke-related molecule also can be detected by measuring the cellular level of ischemic stroke-related nucleic acid molecule-specific mRNA. mRNA can be measured using techniques well known in the art, including for instance Northern analysis, RT-PCR and mRNA in situ hybridization. Details of mRNA analysis procedures can be found, for instance, in provided examples and in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
Oligonucleotides specific to ischemic stroke-related sequences can be chemically synthesized using commercially available machines. These oligonucleotides can then be labeled, for example with radioactive isotopes (such as 32P) or with non-radioactive labels such as biotin (Ward and Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-57, 1981) or a fluorophore, and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis. These specific sequences are visualized, for example by methods such as autoradiography or fluorometric (Landegren et al., Science 242:229-37, 1989) or colorimetric reactions (Gebeyehu et al., Nucleic Acids Res. 15:4513-34, 1987).
Nucleic acid molecules isolated from PBMCs can be amplified using routine methods to form nucleic acid amplification products. These nucleic acid amplification products can then be contacted with an oligonucleotide probe that will hybridize under stringent conditions with an ischemic stroke-related nucleic acid. The nucleic acid amplification products which hybridize with the probe are then detected and quantified. The sequence of the oligonucleotide probe can bind specifically to a nucleic acid molecule represented by the sequences listed in Tables 2-5.
This example describes methods that can be used to detect changes in expression of ischemic stroke-related proteins. Ischemic stroke-related protein sequences can be used in methods of evaluating a stroke, for example for determining whether a subject has had an ischemic stroke, determining the severity or likely neurological recovery of a subject who has had an ischemic stroke, and determining a treatment regimen for a subject who has had an ischemic stroke. For such procedures, a biological sample of the subject is assayed for a change in expression (such as an increase or decrease) of any combination of at least four ischemic stroke-related proteins, such as any combination of at least four of those listed in Table 5, at least 150 of those listed in Table 3, or at least 500 of those listed in Table 2. In some examples, protein expression is determined for any combination of at least one gene from each of the four classes of genes listed in Table 5 (such as at least 2 or at least 3 genes from each of the four classes of genes listed in Table 5). In some examples, protein expression is determined for at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146.
Suitable biological samples include samples containing protein obtained from cells of a subject, such as those present in peripheral blood. A change in the amount of four or more ischemic stroke-related proteins in a subject, such as an increase in four or more ischemic stroke-related proteins listed in Table 5, can indicate that the subject has suffered an ischemic stroke.
The determination of increased or decreased ischemic stroke-related protein levels, in comparison to such expression in a normal subject (such as a subject who has not previously had an ischemic stroke), is an alternative or supplemental approach to the direct determination of the expression level of ischemic stroke-related nucleic acid sequences by the methods outlined above. The availability of antibodies specific to ischemic stroke-related protein(s) will facilitate the detection and quantitation of ischemic stroke-related protein(s) by one of a number of immunoassay methods that are well known in the art, such as those presented in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). Methods of constructing such antibodies are known in the art. Any standard immunoassay format (such as ELISA, Western blot, or RIA assay) can be used to measure ischemic stroke-related protein levels. A comparison to wild-type (normal) ischemic stroke-related protein levels and an increase or decrease in ischemic stroke-related polypeptide levels (such as an increase in any combination of at least 4 proteins listed in Table 5 or a decrease in any combination of at least 4 proteins listed in Tables 2-4 with a negative t-statistic) is indicative of ischemic stroke. Immunohistochemical techniques can also be utilized for ischemic stroke-related protein detection and quantification. For example, a tissue sample can be obtained from a subject, and a section stained for the presence of an ischemic stroke-related protein using the appropriate ischemic stroke-related protein specific binding agents and any standard detection system (such as one that includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in Bancroft and Stevens (Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
For the purposes of quantitating ischemic stroke-related proteins, a biological sample of the subject that includes cellular proteins can be used. Quantitation of an ischemic stroke-related protein can be achieved by immunoassay and the amount compared to levels of the protein found in cells from a subject who has not had an ischemic stroke. A significant increase in the amount of four or more ischemic stroke-related proteins in the cells of a subject compared to the amount of the same ischemic stroke-related protein found in normal human cells is usually at least 2-fold, at least 3-fold, at least 4-fold or greater difference. Substantial overexpression of four or more ischemic stroke-related protein(s) can be indicative of an ischemic stroke. Similarly, a significant decrease in the amount of four or more ischemic stroke-related proteins in the cells of a subject compared to the amount of the same ischemic stroke-related protein found in normal human cells is usually at least 2-fold, at least 3-fold, at least 4-fold or greater difference. Substantial underexpression of four or more ischemic stroke-related protein(s) can be indicative of an ischemic stroke or poor prognosis.
An alternative method of evaluating a stroke is to quantitate the level of four or more ischemia-related proteins in a subject, for instance in the cells of the subject. This diagnostic tool is useful for detecting reduced or increased levels of ischemia-related proteins, for instance, though specific techniques can be used to detect changes in the size of proteins, for instance. Localization or coordinated expression (temporally or spatially) of ischemia-related proteins can also be examined using well known techniques.
Kits are provided for evaluating a stroke, for example for determining whether a subject has had an ischemic stroke, determining the severity or likely neurological recovery of a subject who has had an ischemic stroke, and determining a treatment regimen for a subject who has had an ischemic stroke (such as kits containing ischemic stroke detection arrays). Kits are also provided that contain the reagents need to detect hybridization complexes formed between oligonucleotides on an array and ischemic stroke-related nucleic acid molecules obtained from a subject, or between proteins or antibodies on an array and proteins obtained from a subject suspected of having had (or known to have had) an ischemic stroke. These kits can each include instructions, for instance instructions that provide calibration curves or charts to compare with the determined (such as experimentally measured) values. The disclosed kits can include reagents needed to determine gene copy number (genomic amplification or deletion), such as probes or primers specific for an ischemia-related nucleic acid sequence.
Kits are provided to determine the level (or relative level) of expression or of any combination of four or more ischemic stroke-related nucleic acids (such as mRNA) or ischemic stroke-related proteins (such as kits containing nucleic acid probes, proteins, antibodies, or other ischemic stroke-related protein specific binding agents) listed in Tables 2-5.
Kits are provided that permit detection of ischemic stroke-related mRNA expression levels (including over- or under-expression, in comparison to the expression level in a control sample). Such kits include an appropriate amount of one or more of the oligonucleotide primers for use in, for instance, reverse transcription PCR reactions, and can also include reagents necessary to carry out RT-PCR or other in vitro amplification reactions, including, for instance, RNA sample preparation reagents (such as an RNAse inhibitor), appropriate buffers (such as polymerase buffer), salts (such as magnesium chloride), and deoxyribonucleotides (dNTPs).
In some examples, kits are provided with the reagents needed to perform quantitative or semi-quantitative Northern analysis of ischemic stroke-related mRNA. Such kits can include at least four ischemic stroke-related sequence-specific oligonucleotides for use as probes. Oligonucleotides can be labeled, for example with a radioactive isotope, enzyme substrate, co-factor, ligand, chemiluminescent or fluorescent agent, hapten, or enzyme.
Kits are provided that permit detection of ischemic stroke-related genomic amplification or deletion. Nucleotide sequences encoding an ischemic stroke-related protein, and fragments thereof, can be supplied in the form of a kit for use in detection of ischemic stroke-related genomic amplification/deletion or diagnosis of an ischemic stroke, progression of an ischemic stroke, or therapy assessment for subjects who have suffered an ischemic stroke. In examples of such a kit, an appropriate amount of one or more oligonucleotide primers specific for an ischemic stroke-related-sequence (such as those listed in Table 1) is provided in one or more containers. The oligonucleotide primers can be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. In some applications, pairs of primers are provided in pre-measured single use amounts in individual, typically disposable, tubes, or equivalent containers. With such an arrangement, the sample to be tested for the presence of ischemic stroke-related genomic amplification/deletion can be added to the individual tubes and in vitro amplification carried out directly.
The amount of each primer supplied in the kit can be any amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided is likely an amount sufficient to prime several in vitro amplification reactions. Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines can be found in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
A kit can include more than two primers to facilitate the in vitro amplification of ischemic stroke-related genomic sequences, such as those listed in Tables 2-5, or the 5′ or 3′ flanking region thereof.
In some examples, kits also include the reagents needed to perform in vitro amplification reactions, such as DNA sample preparation reagents, appropriate buffers (for example polymerase buffer), salts (for example magnesium chloride), and deoxyribonucleotides (dNTPs). Written instructions can also be included. Kits can further include labeled or unlabeled oligonucleotide probes to detect the in vitro amplified sequences. The appropriate sequences for such a probe will be any sequence that falls between the annealing sites of two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the in vitro amplification reaction (if it is present in the sample).
One or more control sequences can be included in the kit for use in the in vitro amplification reactions. The design of appropriate positive and negative control sequences is well known to one of ordinary skill in the art.
In particular examples, a kit includes an array with oligonucleotides (or antibodies) that recognize any combination of at least four ischemic stroke-related sequences, such as any combination of at least four of those listed in Table 5, at least 22 of those listed in Table 5, at least 150 of those listed in Table 3, or at least 500 of those listed in Table 2. In one example, the array includes oligonucleotides (or antibodies) that recognize at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 of the following: CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146. For example, the array can include oligonucleotides (or antibodies) that recognize at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146.
In some examples, the array includes agents (such as oligonucleotides, proteins, or antibodies) that can recognize any combination of at least one gene (or protein) from each class listed in Table 5 (such as at least 2 or at least 3 genes (or proteins) from each class). The array can include other oligonucleotides, for example to serve as negative or positive controls. The oligonucleotides that recognize ischemic stroke-related and control sequences can be on the same array, or on different arrays. Particular arrays are disclosed in Examples 7-9.
Kits are also provided for the detection of ischemic stroke-related protein expression, for instance increased expression of any combination of at least 4 proteins listed in Table 5. Such kits include one or more ischemic stroke-related proteins (full-length, fragments, or fusions) or specific binding agent (such as a polyclonal or monoclonal antibody or antibody fragment), and can include at least one control. The ischemic stroke-related protein specific binding agent and control can be contained in separate containers. The kits can also include agents for detecting ischemic stroke-related protein:agent complexes, for instance the agent can be detectably labeled. If the detectable agent is not labeled, it can be detected by second antibodies or protein A, for example, either of both of which also can be provided in some kits in one or more separate containers. Such techniques are well known.
Additional components in some kits include instructions for carrying out the assay. Instructions permit the tester to determine whether ischemic stroke-linked expression levels are elevated, reduced, or unchanged in comparison to a control sample. Reaction vessels and auxiliary reagents such as chromogens, buffers, enzymes, etc. can also be included in the kits.
With the disclosure of many ischemic stroke-related molecules (as represented for instance by those listed in Tables 2-5), gene expression profiles that provide information on evaluating a stroke, for example for determining whether a subject has had an ischemic stroke, determining the severity or likely neurological recovery of a subject who has had an ischemic stroke, and determining a treatment regimen for a subject who has had an ischemic stroke, are now enabled.
Ischemic stroke-related expression profiles include the distinct and identifiable pattern of expression (or level) of sets of ischemic stroke-related genes, for instance a pattern of increased and decreased expression of a defined set of genes, or molecules that can be correlated to such genes, such as mRNA levels or protein levels or activities. The set of molecules in a particular profile can include any combination of at least four of the sequences listed in any of Tables 2-5.
Another set of molecules that could be used in a profile include any combination of at least four sequences listed in Table 5, each of which is overexpressed following an ischemic stroke. For example, an ischemic stroke-related gene expression profile can include one sequence from each of the following four classes of genes: white blood cell activation and differentiation genes, genes related to hypoxia, genes involved in vascular repair, and genes related to a specific PBMC response to the altered cerebral microenvironment. In another example, the molecules included in the profile include at least CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, or any one of these.
Yet another example of a set of molecules that could be used in a profile would include any combination of at least 150 of the sequences listed in Table 3, whose expression is upregulated or downregulated following an ischemic stroke. In a particular example, a set of molecules that could be used in a profile would include any combination of at least 500 of the sequences listed in Table 2, whose expression is upregulated or downregulated following an ischemic stroke.
Particular profiles can be specific for a particular stage or age of normal tissue (such as PMBCs). Thus, gene expression profiles can be established for a pre-ischemic stroke tissue (such as normal tissue not subjected to an ischemic challenge or preconditioning) or an ischemic challenged tissue. Each of these profiles includes information on the expression level of at least four or more genes whose expression is altered following an ischemic stroke. Such information can include relative as well as absolute expression levels of specific genes. Likewise, the value measured can be the relative or absolute level of protein expression or protein activity, which can be correlated with a “gene expression level.” Results from the gene expression profiles of an individual subject can be viewed in the context of a test sample compared to a baseline or control sample fingerprint/profile.
The levels of molecules that make up a gene expression profile can be measured in any of various known ways, which may be specific for the type of molecule being measured. Thus, nucleic acid levels (such as direct gene expression levels, such as the level of mRNA expression) can be measured using specific nucleic acid hybridization reactions. Protein levels can be measured using standard protein assays, using immunologic-based assays (such as ELISAs and related techniques), or using activity assays. Examples for measuring nucleic acid and protein levels are provided herein; other methods are well known to those of ordinary skill in the art.
Examples of ischemia-related gene expression profiles can be in array format, such as a nucleotide (such as polynucleotide) or protein array or microarray. The use of arrays to determine the presence and/or level of a collection of biological macromolecules is now well known (see, for example, methods described in published PCT application number WO 99/48916, describing hypoxia-related gene expression arrays). In array-based measurement methods, an array can be contacted with polynucleotides (in the case of a nucleic acid-based array) or polypeptides (in the case of a protein-based array) from a sample from a subject. The amount or position of binding of the subject's polynucleotides or polypeptides then can be determined, for instance to produce a gene expression profile for that subject. Such gene expression profile can be compared to another gene expression profile, for instance a control gene expression profile from a subject known to have suffered a stroke, or known to not have suffered a stroke. Such a method could be used to determine whether a subject had an ischemic stroke or determine the prognosis of a subject who had an ischemic stroke. In addition, the subject's gene expression profile can be correlated with one or more appropriate treatments, which can be correlated with a control (or set of control) expression profiles for levels of ischemia, for instance.
Prior to performing any assays to identify agents that alter the activity (such as the expression) of an ischemic stroke-related molecule, rapid screening assays can be used to screen a large number of agents to determine if they bind to an ischemic stroke-related protein.
Rapid screening assays for detecting binding to HIV proteins have been disclosed, for example in U.S. Pat. No. 5,230,998, which is incorporated by reference. Briefly, an ischemic stroke-related protein (such as one or more of those listed in Tables 2-5) is incubated with a first antibody capable of binding to the protein, and incubated with one or more test agents. Excess unbound first antibody is washed and removed, and antibody bound to the ischemic stroke-related protein is detected by adding a second labeled antibody which binds the first antibody. Excess unbound second antibody is then removed, and the amount of detectable label is quantitated. The effect of the binding is then determined in percentages by the formula: (quantity of the label in the absence of the test agent)−(quantity of the label in the presence of the test agent /quantity of the label in the absence of the test agent)×100.
Agents that have a high binding affinity to the ischemic stroke-related protein can then be used in other assays more specifically designed to determine the activity (such as the expression) of an ischemic stroke-related molecule.
This example describes particular in vitro methods that can be used to screen test agents for their ability to alter the activity of an ischemic stroke-related molecule. However, the disclosure is not limited to these particular methods. One skilled in the art will appreciate that other in vitro assays could be used.
As disclosed in the Examples above, expression of the disclosed ischemic stroke-related molecules (such as those listed in Tables 2-5) is increased or decreased following an ischemic stroke. Therefore, screening assays can be used to identify and analyze agents that normalize such activity (such as decrease expression/activity of a gene that is increased following an ischemic stroke, increase expression/activity of a gene that is decreased following an ischemic stroke, or combinations thereof), or further enhance the change in activity (such as further decrease expression/activity of a gene that is decreased following an ischemic stroke, or further increase expression/activity of a gene that is increased following an ischemic stroke). For example, it may be desirable to further enhance the change in activity if such a change provides a beneficial effect to the subject or it may be desirable to neutralize the change in activity if such a change provides a harmful effect to the subject.
Agents identified via the disclosed assays can be useful, for example, in decreasing one or more symptoms associated with stroke, such as a decrease of at least about 10%, at least about 20%, at least about 50%, or even at least about 90%. Once identified, test agents found to alter the activity of an ischemic stroke-related molecule can be formulated in therapeutic products (or even prophylactic products) in pharmaceutically acceptable formulations, and used to treat a subject who has had an ischemic stroke.
Cells (such as at least 50,000 cells) that provide a model what happens in vivo following an ischemic stroke are cultured under hypoxic conditions, hypoglycemic conditions, or combinations thereof. For example, PBMCs can be cultured at 37° C. in hypoxic conditions of 94% N2, 5% CO2, and 1% O2, for at least 1 hour, such as 4 hours or 24 hours. In another example, PBMCs are cultured at 37° C. in the absence of added glucose for at least 1 hour, such as 4 hours or 24 hours. In yet another example, PBMCs are cultured at 37° C. in at 94% N2, 5% CO2, and 1% 02 in the absence of added glucose for at least 1 hour, such as 4 hours or 24 hours.
Simultaneous to incubation in the hypoxic or hypoglycemic conditions, or at a time later, one or more test agents are incubated with the cells under conditions sufficient for the test agent to have the desired effect on the cell, for example to alter (such as normalize) the activity of a ischemic stroke-related molecule. In one example, the agent is added at least 30 minutes after culturing the cells in the hypoxic or hypoglycemic conditions, such as at least 1 hour, at least 2 hours, at least 6 hours, or at least 24 hours after culturing the cells in the hypoxic or hypoglycemic conditions.
To determine the effect of the test agents on the activity of one or more ischemic stroke-related molecules, RNA can be isolated from the PBMCs and labeled (see Examples 1 and 2). The labeled RNA is exposed to an array containing one or more nucleic acid molecules (such as a primer or probe) that can specifically hybridize to one or more ischemic stroke-related genes, such at least 1, at least 2, or at least 3 of those listed in Tables 2-5 (for example using the methods described herein).
Alternatively, to determine the effect of the test agents on the activity of one or more ischemic stroke-related molecules, proteins are isolated from the PBMCs. The isolated proteins can be analyzed to detect differential expression of one or more ischemic stroke-related proteins, such at least 1, at least 2, or at least 3 of those listed in Tables 2-5, such as using the methods described in Example 9
This example describes particular in vivo methods that can be used to screen test agents for their ability to alter the activity of an ischemic stroke-related molecule. However, the disclosure is not limited to these particular methods. One skilled in the art will appreciate that other in vivo assays could be used (such as other mammals or other means of inducing an ischemic stroke).
A mammal is exposed to conditions that induce an ischemic stroke. In a particular example, an ischemic stroke is induced in a mouse by occlusion of the middle cerebral artery (MCA) under anesthesia (for example 1 mL/kg of a mixture of ketamine (75 mg/mL) and xylazine (5 mg/mL)). The mouse is anesthetized and a U-shape incision made between the left ear and left eye. The top and back segments of the temporal muscle are transected, and the skull exposed by retraction of the temporal muscle. A small opening (1 to 2 mm in diameter) is made in the region over the MCA with saline superfusion to prevent heat injury. The meninges can be removed, and the MCA occluded by ligation, for example with 10-0 nylon thread (Ethylon). Occlusion of the MCA can be persistent (for example by transecting the MCA distally to the ligation point), or reversible, for example by occluding for a finite period of time, such as at least 10 minutes, at least 30 minutes, or at least 60 minutes. Alternatively or in addition, the mouse is exposed hypoxic conditions, such as 8-11% oxygen for 2 hours.
Simultaneous to inducing the ischemic stroke, or at a time later, one or more test agents are administered to the subject under conditions sufficient for the test agent to have the desired effect on the subject. Any appropriate method of administration can be used, such as intravenous, intramuscular, or transdermal. In one example, the agent is added at least 30 minutes after the ischemic stroke, such as at least 1 hour, at least 2 hours, at least 6 hours, or at least 24 hours after the ischemic stroke.
The effect of the test agents on the activity of one or more ischemic stroke-related molecules can be determined using methods described in Example 15. For example, PBMCs can be isolated from the subject following exposure to the test agent. RNA or proteins isolated from the PBMCs can be analyzed to determine the activity of one or more ischemic stroke-related molecules.
This example describes methods that can be used to further evaluate test agents that alter the activity of an ischemic stroke-related molecule, such as those identified using the methods described in Examples 15 and 16. For example, effective doses of the test agents, and the ability of the agent to treat an ischemic stroke can be determined in vitro or in vivo.
Cells (such as 20,000 to 500,000 cells) are exposed to conditions that mimic an ischemic stroke, such as hypoxic or hypoglycemic conditions (or both), and the incubation continued for at least 1 hour (such as at least 4 hours or at least 24 hours). The test agent can be applied to the cells before, during, or after mimicking an ischemic stroke. In some examples, several different doses of the potential therapeutic agent are administered, to identify optimal dose ranges. For example, milligram, microgram, and nanogram concentrations can be used. Subsequently, assays are conducted to determine the activity of one or more ischemic stroke-related molecules.
The ability of an agent, such as those identified using the methods provide above, to treat an ischemic stroke, can be assessed in animal models. Several methods of inducing an ischemic stroke in a mammal are known, and particular examples are provided herein. Mammals of any species, including, but not limited to, mice, rats, rabbits, dogs, guinea pigs, pigs, micro-pigs, goats, and non-human primates, such as baboons, monkeys, and chimpanzees, can be used to generate an animal model of ischemic stroke. Such animal models can also be used to test agents for an ability to ameliorate symptoms associated with ischemic stroke. In addition, such animal models can be used to determine the LD50 and the ED50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential agents.
An ischemic stroke is induced in the mammal, and one or more test agents identified in the examples above administered. The amount of test agent administered can be determined by skilled practitioners. In some examples, several different doses of the potential therapeutic agent can be administered to different test subjects, to identify optimal dose ranges. The therapeutic agent can be administered before, during, or after inducing the ischemic stroke. Subsequent to the treatment, animals are observed for one or more symptoms associated with ischemic stroke. A decrease in the development of symptoms associated with ischemic stroke in the presence of the test agent provides evidence that the test agent is a therapeutic agent that can be used to decrease or even inhibit ischemic stroke in a subject.
This example describes particular changes in expression, such as gene or protein expression, that are associated with ischemic stroke. Although particular ischemic stroke-related molecules are listed in this example, one skilled in the art will appreciated that other molecules can be used based on the teachings in this disclosure.
In particular examples detecting differential expression includes detecting differences in expression (such as an increase, decrease, or both). The method can further include determining the magnitude of the difference in expression, wherein the magnitude of the change is associated with ischemic stroke. Particular examples of ischemic stroke-related molecules that are differentially expressed in association with the diagnosis of an ischemic stroke, and their direction of change (upregulated or downregulated), and the magnitude of the change (as expressed as a percent, t-statistic, and fold change) are provided in Table 10.
Therefore, CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146 are upregulated by a magnitude of at least 50%, at least 4-fold or have a t-statistic of at least 4. That is, CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146 are upregulated by an amount associated with ischemic stroke, for example at least 50% or at least 4-fold (or have a t-statistic of at least 4). In addition, intercellular adhesion molecule 2, protein kinase D2, GATA binding protein 3, hypothetical protein FLJ20257, and protein kinase C, theta are downregulated by a magnitude of at least 50%, at least 4-fold or have a t-statistic of no more than −5. That is, intercellular adhesion molecule 2, protein kinase D2, GATA binding protein 3, hypothetical protein FLJ20257, and protein kinase C, theta are downregulated by an amount associated with ischemic stroke, for example at least 50% or at least 4-fold (or have a t-statistic of no more than −5).
One example of a pattern of expression of proteins that have been found to be associated with ischemic stroke, such as upregulation of CD163; hypothetical protein FLJ22662 Laminin A motif; and BST-1, wherein the magnitude of change is at least 4-fold for each of CD163; hypothetical protein
FLJ22662 Laminin A motif; and BST-1. Another example of a pattern of expression of proteins that have been found to be associated with ischemic stroke is as upregulation of CD163; hypothetical protein FLJ22662 Laminin A motif; BST-1; FcγRI; baculoviral IAP repeat-containing protein 1; and KIAA0146, for example wherein the magnitude of change is at least 4-fold for each of these proteins.
In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that the illustrated embodiment is only a preferred example of the invention and should not be taken as a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
This is a divisional of U.S. application Ser. No. 11/155,835 filed Jun. 17, 2005, now U.S. Pat. No. 7,749,700 issued Jul. 6, 2010, which is a continuation-in-part of PCT/US2005/018744 filed May 27, 2005, which claims priority to U.S. Provisional Application No. 60/575,279 filed May 27, 2004, all herein incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60575279 | May 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11155835 | Jun 2005 | US |
| Child | 12829229 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2005/018744 | May 2005 | US |
| Child | 11155835 | US |
