WO2012016030A2 - Inflammation detection - Google Patents

Abstract

The present invention relates to methods and reagents for detecting and monitoring inflammation and injury in a subject by determining the expression levels of groups of genes.

Description

INFLAMMATION DETECTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No. 61/432,387, filed on January 13, 2011, and U.S. Provisional Application No. 61/368,532, filed on July 28, 2010. The contents of the applications are incorporated herein by reference in their entireties.

GOVERNMENT INTERESTS

The invention disclosed herein was made, at least in part, with Government support under Grant No. NIH GM 34695 from the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention. FIELD OF THE INVENTION

This invention relates to reagents and methods for detecting inflammation in a subject.

BACKGROUND OF THE INVENTION

Inflammation is a complex biological response of the body to harmful stimuli, such as pathogens, damaged cells, or irritants. It is involved in various inflammatory disorders. Inflammatory disorders, characterized by the abnormal activation and subsequent migration of leukocytes or white blood cells to affected areas of the body, encompass a wide range of ailments that affect the lives of millions of people throughout the world. Few tests exist that reliably diagnose or monitor the progress of inflammation and the disorders. Thus, there is a need for reagents and methods for detecting inflammation.

Circulating leukocytes play a central role in host immunity, and are a major source of inflammatory mediators released in response to exposure to pathogen-associated molecular pattern(s) (PAMPs), such as endotoxin. Gene expression profiling of human peripheral blood leukocytes (PBL) or mononuclear cells have revealed robust gene expression changes that are detectable within 2 hours of an in vivo endotoxin challenge. The acute phase of systemic inflammation is associated with the release of numerous cytokines and inflammatory mediators, as well as global changes in gene expression in PBL. Yet, changes in cytokines are significantly less robust, and hence difficult to establish, during conditions of low-grade inflammation. Thus, there also remains a need for an improved method of identifying the presence of inflammation and for monitoring a patient' s response. SUMMARY OF INVENTION

This invention is based, at least in part, on the unexpected discovery of a group of genes that exhibit similar expression trends in PBL derived from trauma patients and from subjects challenged with endotoxin, which induced acute inflammation via the Toll-like receptor4 (TLR4) pathway. As disclosed herein, the TLR4-induced transcription patterns elicited in humans exposed to endotoxin parallel gene expression patterns observed in trauma patients with initial non-infectious injury.

Accordingly, in one aspect, this invention features a method for determining whether a subject has, or is at risk of having, an inflammatory disorder, e.g., sepsis or a systemic inflammatory response syndrome. The method includes, among others, steps of obtaining from the subject a sample and determining in the sample the expression levels of a plurality of genes. Each of the genes is selected from (i) a first panel of up-regulated TLR4 and Injury Responsive (TIR) genes, (ii) a second panel of down-regulated TIR genes, or (iii) a third panel of core genes. The subject is determined to have, or to be at risk of having, the disorder if: (a) the expression level of each gene selected from the first panel is above a first predetermined reference value; (b) the expression level of each gene selected from the second panel is below a second predetermined reference value; or (c) the expression level of each gene selected from the third panel is below a third predetermined reference value. The sample can be a blood sample or any other suitable sample that contains leukocytes. The first, second, or third predetermined reference value can be obtained from a control subject that does not have the disorder.

The afore-mentioned "TLR4 and Injury Responsive (TIR) genes" refers to genes that exhibit persisted differential expression (i.e., up-regulated or down-regulated) in response to TLR4-induced systemic inflammation and/or injury, which can be determined by the method described in Examples 1 and 2 below. This group of genes includes the 449 genes listed in Table 3. These 449 genes include (a) a first panel of 176 up-regulated TIR genes whose expression levels in a healthy subject increase (i.e., up-regulated) in response to an endotoxin challenge and (b) a second panel of 273 down-regulated TIR genes whose expression levels in a healthy subject decrease (i.e., done-regulated) in response to an endotoxin challenge.

In one embodiment, each gene for practicing the above-described method is selected from the third panel of core genes. This panel of core genes can include the Cryl, Cry2, Per3, Clock, Rora, Rev, CSNKle, and CDK4 genes. In one example, this panel consists of the Cryl, Cryl, Per 3, Clock, Rora, Rev, CSNKle, and CDK4 genes. In a second aspect, the invention features a method for determining a prognosis of an inflammatory disorder (e.g., sepsis or a systemic inflammatory response syndrome) in a subject that has received an injury. The method includes, among others, steps for obtaining from the subject a sample and for determining in the sample the magnitude of a change in the expression level of one or more genes. Each of the genes is selected from (i) the first panel of up-regulated TIR genes, (ii) the second panel of down-regulated TIR genes, (iii) the third panel of core genes, or (iv) a fourth panel of reversible responsive genes. The "reversible responsive genes" refers to a panel of genes that show differential expression in response to TLR4- induced systemic inflammation and/or injury, but reverse their expression trends within a period of time, such as 9-12 days. Examples of these genes include the 150 genes listed in Table 4. The just-mentioned magnitude (i.e., extent or degree) of the change is indicative of the prognosis of the subject.

The prognosis method can further include a step of comparing the magnitude to a predetermined magnitude reference value whereby the subject is determined to have a good prognosis if the magnitude is below the predetermined reference value. The predetermined magnitude reference value can be obtained from a patient that has the disorder. Alternatively, it can be obtained from the subject at a time after receiving the injury, but before the above- mentioned sample is obtained. In other words, a decrease in the magnitude over time indicates a good prognosis. The sample can be a blood sample or any other suitable sample that contains leukocytes.

In one embodiment, the one or more genes for practicing the prognosis method are selected from the fourth panel. In one example, the sample can be obtained from the subject within 12 days after receiving the injury or after the onset of the disorder. Preferably, the sample is obtained from the subject within, e.g., 9-12 days after receiving the injury or after the onset of the disorder. In another embodiment, the one or more genes are selected from the first, second, or third panel. In this embodiment, the sample can be obtained from the subject, e.g., 12 or more days after receiving the injury or after the onset of the disorder.

In some embodiments, one or more panels of glycolysis genes, RPL/KPS genes, EIF/EEF, and HNRNP genes, which are listed in Table 3 or 4 and further described in the examples below, can be used to practice the above-described methods.

In a third aspect, the invention features an array for determining whether a subject has, or is at risk of having, an inflammatory disorder or for determining a prognosis of an inflammatory disorder in a patient that has received an injury. The array includes a support having a plurality of unique locations, and any combination of (i) at least one nucleic acid having a sequence that is complementary to the sequence of a gene selected from the first panel of up-regulated TIR genes, (ii) at least one nucleic acid having a sequence that is complementary to the sequence of a gene selected from the second panel of down-regulated TIR genes, (iii) at least one nucleic acids having a sequence that is complementary to the sequence of a gene selected from the third panel of core genes, and (iv) at least one nucleic acid having a sequence that is complementary to the sequence of a gene selected from the fourth panel of reversible responsive genes. Each type of nucleic acid is immobilized or attached to a unique location of the support.

In one example, the array has at least eight (e.g., 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,

60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, or 1000) types of unique nucleic acids and corresponding unique locations on the support. The unique locations can be selected from the group consisting of beads, spheres and optical fibers. The support can contain a material selected from the group consisting of glass, coated glass, silicon, porous silicon, nylon, ceramic, and plastic.

In a fourth aspect, the invention features a kit. The kit contains (1) a probe having a nucleic acid sequence that is complementary to the sequence of a gene selected from the above-mentioned first panel of up-regulated TIR genes, second panel of down-regulated TIR genes, third panel of core genes, or fourth panel of reversible responsive genes, or (2) a pair of PCR primers for amplifying a mRNA of the selected gene. The kit can further contain reagents (e.g., buffers, substrates, and enzymes) for performing hybridization or PCR.

In one embodiment, the gene is selected from the third panel of core genes, comprising or consisting of the Cryl, Cry2, Per3, Clock, Rora, Rev, CSNKle, and CDK4 genes. In this case, the kit can be one having eight probes that are complementary to sequences of the Cryl, Cry2, Per3, Clock, Rora, Rev, CSNKle, and CDK4 genes, respectively, or one of eight pairs of primers for amplifying mRNAs of the Cryl, Cry2, Per3, Clock, Rora, Rev, CSNKle, and CDK4 genes, respectively. In another embodiment, the kit contains one or more of the above- mentioned arrays.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-D are diagrams showing TLR4 and injury responsive (TIR) genes selection criteria. (A) Genes that were significantly differentially expressed in PBL obtained from subjects challenged with in vivo endotoxin (Endo) for 6 hours (2 ng/kg), subjects infused with Cortisol (Cort) ^g/kg/min) for 12 hours starting 6 hours before endotoxin administration (Cort+Endo), or from trauma patients PBL obtained within the initial 5 days after ICU admission, as compared to baseline, were identified. The Venn diagram identifies the genes that are common between groups. Nine hundred thirty seven (937) genes were common to all three groups. (B) Scatter plot analysis comparing Group 1 genes expression trends between the indicated groups. (C) Genes that were significantly differentially expressed in trauma patients PBL obtained within 9-12 days after ICU admission as compared to baseline were identified. (D) Four hundred and forty-nine genes were differentially expressed in both in vivo endotoxin challenged PBL and in PBL obtained from trauma patients over a period of 1-12 days after admission.

FIG. 2 is a diagram showing TIR genes pathway analysis. To determine the putative biological role of the TIR genes, the expression data were analyzed through the use of Ingenuity Pathway Analysis (Ingenuity® systems, www.ingenuity.com). The top ranking module shown in this figure includes 99 TIR genes. Myc, depicted on the lower right, is the focal point for the most densely populated node that includes numerous RPL/RPS genes.

FIGs. 3A-D are a set of diagrams showing expression of clock genes (Bmall, Clock,

Cryl, Cry2, Per3 and Rora) as determined by qPCR in PBLs from 4 control and 3 surgical ICU patients; FIG. 3A shows the mean fold change in gene expression observed in the PBLs, where error bars are SEM. Two blood samples, referred to as first (days 0-5) and second blood draw (days 9-12), were obtained from each patient at a one-week interval; FIGs. 3B-D show the fold change in the expression for each of the patients.

FIGs. 4A-D are diagrams showing expression of circadian clock gene in PBL obtained from human subjects challenged with endotoxin or saline at 9 AM.

FIG. 5 is a set of diagrams showing circadian clock gene expression in PBL obtained from human subjects challenged with endotoxin at 9 PM.

FIG. 6 is a set of diagram showing circadian clock gene expression in PBL, neutrophils, and monocytes obtained from human subjects challenged with endotoxin at 9 AM.

FIGs. 7A and 8B are diagrams showing that endotoxin does not alter melatonin's diurnal rhythmicity. FIGs. 8A-E are diagrams showing cross-correlation analyses of clock gene expression in human PBL challenged with endotoxin or saline at 9 AM and/or 9 PM.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to reagents and methods for detecting inflammation in a subject. This invention is based, at least in part, on the unexpected discovery that the TLR4-induced transcription patterns elicited in humans exposed to in vivo endotoxin parallel gene expression patterns observed in trauma patients with initial non-infectious injury. As described in the examples below, several groups of genes that are differentially expressed in PBL upon endotoxin challenge or injury were identified. These genes can be used for determining whether a subject has, or is at risk of having, an inflammatory disorder or for determining a prognosis of an inflammatory disorder in a subject that has received an injury.

Genes Involved in TLR4 Signaling

A large number of genes identified are involved in TLR4 signaling. Based on their expression patterns, these genes can be divided into two groups.

i. TLR4 and Injury Responsive Genes

Using sequential selection criteria of gene expression data described below, 449 genes that are significantly differentially expressed (both p<0.05 and > 1.2 fold-change) in PBL derived from human subjects during the peak of systemic inflammatory responses induced by in vivo endotoxin, as well as in PBL obtained from trauma patients at 1-12 days after admission were identified. These 449 genes exhibited similar expression trends in PBL in both endotoxin-challenged subjects and trauma patients. While these changes in TLR4 induced gene expression are short-lived in LPS challenged subjects, the patterns observed for the 449 genes after injury persist for up to 12 days after trauma. They do not reverse their expression trends in trauma patients by 9 to 12 days post-trauma. These genes are referred TLR4 and injury responsive genes ("TIR" genes) and listed in Table 3 below.

Included in this group are multiple down-regulated genes that are associated with the translational apparatus, as well as several up-regulated genes, including those encoding proteins exhibiting a key role in glycolysis. Specifically, among these genes, two functional modules were identified. The first module includes more than 50 suppressed genes that encode ribosomal proteins or translation regulators. The second module includes up-regulated genes encoding key enzymes associated with glycolysis. These observations identify common TLR4/injury induced transcriptional themes that exist in PBL during systemic inflammation and trauma. Other studies in animal models have highlighted that TLR4 signaling is initiated not only by pathogen-associated molecular patterns (PAMPs), but also by damage-associated molecular patterns (DAMPs) that are released by host tissues when exposed to more extreme stress conditions, such as injury and infection. High-mobility group box 1 (HMGBl), and heat shock proteins (HSP) HSP-70 and HSP-90, are examples of DAMPs that signal through TLR4. In addition, there is evidence that cellular reactive oxygen species (ROS) may also engage TLR4 and activate TLR-dependent signaling events. Collectively, these data imply that endogenous DAMPs and ROS, as well as endotoxin or other PAMPs, have the capacity to initiate common, TLR4-related signaling cascades.

ii. Reversible Responsive Genes

Genes of this group are differentially expressed in PBL obtained from endotoxin challenged subjects and in trauma patients within the first 5 days of admission as compared to baseline reference values of normal subjects. It has been found that approximately 150 of these genes reversed their expression trends in all trauma patients by 9 to 12 days post- trauma. These genes demonstrated an early recovery pattern following endotoxin challenge.

Thus, changes in expression levels and/or trends of this gene group (or a subset of it) in peripheral blood immune cells, which can be determined by either quantitative real-time PCR or gene arrays, can provide an early indication of recovery or lack thereof. For example, a further decline or persistently-altered gene expression levels indicate a poor prognosis, i.e., lack of improvement or health decline. Accordingly, these genes allow one to assess post- inflammation recovery of injury, trauma, and/or other critical and/or inflammatory illnesses. The analysis of this select group of genes or a subset thereof indicates outcomes of the conditions.

Circadian Clock/Core Genes

Another group of genes that are suppressed in PBL from both endotoxin challenged subjects and ICU patients are circadian clock genes.

As disclosed herein, endotoxin causes profound suppression of many clock genes in PBL with a nadir being reached at 3-6 hours post-infusion. The suppressed expression persisted for at least 17 hours, while plasma melatonin's rhythmicity remained intact. In addition, the endotoxin-induced decrease in circadian gene expression was evident during two differing clock phases. These studies establish that endotoxin is a potent, acute entrainer of the circadian clock gene network in PBL in humans. For instance, the results described in Example 3 below demonstrate severe misalignment of central circadian indicators from the peripheral clock in PBL during the acute phase of systemic inflammation. Furthermore, the circadian rhythmicity of melatonin secretion is suppressed in severely ill patients, whereas in rodents, absence of circadian cues during recovery from sepsis impairs survival. The realignment of the central and peripheral clocks constitutes a previously unappreciated key factor affecting recovery.

The circadian clock genes, which are collectively suppressed in peripheral blood immune cells during the acute phase of systemic inflammation, remain suppressed by the time the level of other inflammatory indicators has returned to normal. The genes include the Cryl, Cry2, Per3, Clock, Rora, Rev, and CSNKle, all of which have been implicated in the regulation of circadian clocks. This group of seven clock genes and the CDK4 gene are referred to as "core genes." The core genes expression levels in peripheral blood immune cells can be determined by either quantitative real-time PCR or gene arrays. This combination of altered core genes expression level is also indicative of inflammation.

Diagnosis and Prognosis Methods

The above-describe genes, related kits or arrays, can be used in determining whether a subject has, or is at risk of having, an inflammatory disorder. Alternatively, they can be used for determining a prognosis of an inflammatory disorder in a subject that has received an injury.

Accordingly, this invention also provides diagnostic methods. A subject having an inflammatory disorder or prone to it can be determined based on the expression levels, patterns, or profiles of the above-described genes or their products, such as nucleic acids (e.g., mRNA) or polypeptides in a test sample from the subject. In other words, the polypeptide and nucleic acids can be used as markers to indicate the presence or absence of the disorder or the risk of having the disorder. Diagnostic and prognostic assays of the invention include methods for assessing the expression level of the nucleic acids or polypeptides. The methods and kits allow one to detect low-grade, acute, or chronic inflammation. For example, a relative increase in the eight core gene expression levels may be indicative of recovery from the disorder. Conversely, further decline or persistent low expression levels of one or more of the eight core gene may indicate lack of improvement or health decline.

The presence, level, or absence of the nucleic acid or polypeptide in a test sample can be evaluated by obtaining a test sample from a test subject and contacting the test sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA or genomic DNA probe). The "test sample" includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. The level of expression of a gene(s) of interest can be measured in a number of ways, including measuring the mRNA encoded by the gene; measuring the amount of polypeptide encoded by the gene; or measuring the activity of polypeptide encoded by the gene.

Expressed RNA samples can be isolated from biological samples using any of a number of well-known procedures. For example, biological samples can be lysed in a guanidinium-based lysis buffer, optionally containing additional components to stabilize the RNA. In some embodiments, the lysis buffer can contain purified RNAs as controls to monitor recovery and stability of RNA from cell cultures. Examples of such purified RNA templates include the Kanamycin Positive Control RNA from PROMEGA (Madison, WI), and 7.5 kb Poly(A)-Tailed RNA from LIFE TECHNOLOGIES (Rockville, MD). Lysates may be used immediately or stored frozen at, e.g., -80°C.

Optionally, total RNA can be purified from cell lysates (or other types of samples) using silica-based isolation in an automation-compatible, 96-well format, such as the RNEASY purification platform (QIAGEN, Inc., Valencia, CA). Alternatively, RNA is isolated using solid-phase oligo-dT capture using oligo-dT bound to microbeads or cellulose columns. This method has the added advantage of isolating mRNA from genomic DNA and total RNA, and allowing transfer of the mRNA-capture medium directly into the reverse transcriptase reaction. Other RNA isolation methods are contemplated, such as extraction with silica-coated beads or guanidinium. Further methods for RNA isolation and preparation can be devised by one skilled in the art.

The methods of the present invention can also be performed using crude cell lysates, eliminating the need to isolate RNA. RNAse inhibitors are optionally added to the crude samples. When using crude cellular lysates, it should be noted that genomic DNA can contribute one or more copies of a target sequence, e.g., a gene, depending on the sample. In situations in which the target sequence is derived from one or more highly expressed genes, the signal arising from genomic DNA may not be significant. But for genes expressed at low levels, the background can be eliminated by treating the samples with DNAse, or by using primers that target splice junctions for subsequent priming of cDNA or amplification products. For example, one of the two target- specific primers could be designed to span a splice junction, thus excluding DNA as a template. As another example, the two target- specific primers can be designed to flank a splice junction, generating larger PCR products for DNA or unspliced mRNA templates as compared to processed mRNA templates. One skilled in the art could design a variety of specialized priming applications that would facilitate use of crude extracts as samples for the purposes of this invention.

The level of mRNA corresponding to a gene in a cell can be determined both in situ and in vitros. Messenger RNA isolated from a test sample can be used in hybridization or amplification assays that include, Southern or Northern analyses, PCR analyses, and probe arrays. A preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid probe that can hybridize to the mRNA encoded by the gene. The probe can be a full-length nucleic acid or a portion thereof, such as an oligonucleotide of at least 10 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA.

In one format, mRNA (or cDNA prepared from it) is immobilized on a surface and contacted with the probes, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In another format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a gene chip array. A skilled artisan can adapt known mRNA detection methods for detecting the level of an mRNA.

The level of mRNA (or cDNA prepared from it) in a sample encoded by a gene to be examined can be evaluated with nucleic acid amplification, e.g., by standard PCR (U.S. Patent No. 4,683,202), RT-PCR (Bustin S. J Mol Endocrinol. 25: 169-93, 2000), quantitative PCR (Ong Y. et al, Hematology. 7:59-67, 2002), real time PCR (Ginzinger D. Exp Hematol. 30:503-12, 2002), and in situ PCR (Thaker V. Methods Mol Biol. 115:379-402, 1999), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art.

The term "primer" refers to any nucleic acid that is capable of hybridizing at its 3' end to a complementary nucleic acid molecule, and that provides a free 3' hydroxyl terminus which can be extended by a nucleic acid polymerase. As used herein, amplification primers are a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule having the nucleotide sequence flanked by the primers. For in situ methods, a cell or tissue sample can be prepared and immobilized on a support, such as a glass slide, and then contacted with a probe that can hybridize to mRNA. Alternative methods for amplifying nucleic acids corresponding to expressed RNA samples include those described in, e.g., U.S. Patent No. 7,897,750.

In another embodiment, the methods of the invention further include contacting a control sample with a compound or agent capable of detecting the mRNA of a gene and comparing the presence of the mRNA in the control sample with the presence of the RNA in the test sample.

The above-described nucleic acid-based diagnostic methods can provide qualitative and quantitative information to determine whether a subject has or is predisposed to a disease characterized by inflammation, e.g., sepsis.

A variety of methods can be used to determine the level of the polypeptide encoded by a gene disclosed herein. In general, these methods include contacting an agent that selectively binds to the polypeptide, such as an antibody, to evaluate the level of polypeptide in a sample. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can also be used. In a preferred embodiment, the antibody bears a detectable label. The term "label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable. A label may be incorporated into nucleic acids and proteins at any position. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by physically linking a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. For example, an antibody with a rabbit Fc region can be indirectly labeled using a second antibody directed against the rabbit Fc region, wherein the second antibody is coupled to a detectable substance. Examples of detectable substances are

125 provided herein. Appropriate detectable substance or labels include radio isotopes (e.g., I,

131 35 3 32

I, S, H, or P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles by the Quantum Dot Corporation, Palo Alto, CA).

The detection methods can be used to detect a polypeptide in a biological sample in vitro as well as in vivo. In vitro techniques for detection of the polypeptide include ELISAs, immunoprecipitations, immunofluorescence, EIA, RIA, and Western blotting analysis. In vivo techniques for detection of the polypeptide include introducing into a subject a labeled anti- antibody. For example, the antibody can be labeled with a detectable substance as described above. The presence and location of the detectable substance in a subject can be detected by standard imaging techniques.

The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with inflammation. The prognostic assays described herein can be used to determine whether a subject is suitable to be administered with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disorder associated with inflammation. For example, such assays can be used to determine whether a subject can be administered with an immune-suppressant to treat an immune disorder, including those involved in organ/tissue transplantation.

Thus, also provided by this invention is a method of monitoring a treatment for an immune disorder in a subject. For this purpose, gene expression levels of the genes disclosed herein can be determined for test samples from a subject before, during, or after undergoing a treatment. The magnitudes of the changes in the levels as compared to a baseline level are then assessed. A decrease of the magnitudes of the changes after the treatment indicates that the subject can be further treated by the same treatment.

For example, a patient who has received organ or tissue transplantation often faces the problems of organ or tissue rejection. That is, the body has an immune response to an organ or tissue which causes failure of the transplant. To address this problem, organ or tissue transplantation is often accompanied by nonspecific immune suppression therapy to prevent T cell-mediated rejection. However, these immunosuppressants can cause infection, hypertension, cancer, and other undesirable side effects. Therefore, there is a need for monitoring the suppression. To that end, expression levels of the gene disclosed herein can serve as markers for a proper level or degree of treatment, such as immune suppression. A skilled in the art can adjust the amount of a drug and length of treatment based on the levels of gene expression during the course of the treatment.

Information obtained from practice of the above assays is useful in prognostication, identifying progression of, and clinical management of diseases and other deleterious conditions affecting an individual's health status. In preferred embodiments, the foregoing diagnostic assays provide information useful in prognostication, identifying progression of and management of conditions that are characterized by inflammation. The information more specifically assists the clinician in designing chemotherapeutic or other treatment regimes to eradicate such conditions from the body of an afflicted subject, a human.

Arrays

Also provided in the invention is a biochip or array. The biochip/array may contain a solid or semi-solid substrate having an attached probe or plurality of probes described herein. The probes may be capable of hybridizing to a target sequence under stringent hybridization conditions. The probes may be attached at spatially defined address on the substrate. More than one probe per target sequence may be used, with either overlapping probes or probes to different sections of a particular target sequence. The probes may be capable of hybridizing to target sequences associated with a single disorder appreciated by those in the art. The probes may either be synthesized first, with subsequent attachment to the biochip, or may be directly synthesized on the biochip.

"Attached" or "immobilized" as used herein to refer to a nucleic acid (e.g., a probe) and a solid support may mean that the binding between the probe and the solid support is sufficient to be stable under conditions of binding, washing, analysis, and removal. The binding may be covalent or non-covalent. Covalent bonds may be formed directly between the probe and the solid support or may be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Non-covalent binding may be one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non-covalent binding of a biotinylated probe to the streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions.

The solid substrate can be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one detection method. Examples of such substrates include glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyure thanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics. The substrates may allow optical detection without appreciably fluorescing.

The substrate can be planar, although other configurations of substrates may be used as well. For example, probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as flexible foam, including closed cell foams made of particular plastics.

The array/biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the biochip may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the probes may be attached using functional groups on the probes either directly or indirectly using a linker. The probes may be attached to the solid support by either the 5' terminus, 3' terminus, or via an internal nucleotide. The probe may also be attached to the solid support non-covalently. For example, biotinylated oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, probes may be synthesized on the surface using techniques such as photopolymerization and photolithography. Detailed discussion of methods for linking nucleic acids to a support substrate can be found in, e.g., U.S. Patent Nos. 5837832, 6087112, 5215882, 5707807, 5807522, 5958342, 5994076, 6004755, 6048695, 6060240, 6090556, and 6040138.

In some embodiments, an expressed transcript (e.g., a transcript of a gene described herein) is represented in the nucleic acid arrays. In such embodiments, a set of binding sites can include probes with different nucleic acids that are complementary to different sequence segments of the expressed transcript. Examples of such nucleic acids can be of length of 15 to 200 bases, 20 to 100 bases, 25 to 50 bases, 40 to 60 bases. Each probe sequence can also include one or more linker sequences in addition to the sequence that is complementary to its target sequence. A linker sequence is a sequence between the sequence that is complementary to its target sequence and the surface of support. For example, the nucleic acid arrays of the invention can have one probe specific to each target gene or exon. However, if desired, the nucleic acid arrays can contain at least 2, 5, 10, 100, 200, 300, 400, 500 or more probes specific to some expressed transcript (e.g., a transcript of a gene described herein, e.g., in Table 3 or 4). For example, the array may contain probes tiled across the sequence of the longest mRNA isoform of a gene.

Kits

In another aspect, the present invention provides kits embodying the methods, compositions, and systems for analysis of gene expression as described herein.

Such a kit may contain a nucleic acid described herein together with any or all of the following: assay reagents, buffers, probes and/or primers, and sterile saline or another pharmaceutically acceptable emulsion and suspension base. In addition, the kit may include instructional materials containing directions (e.g., protocols) for the practice of the methods described herein. For example, the kit may be a kit for the amplification, detection, identification or quantification of a target mRNA sequence. To that end, the kit may contain a poly(T) primer, a forward primer, a reverse primer, and a probe.

In one example, a kit of the invention includes one or more microarray slides (or alternative microarray format) onto which a plurality of different nucleic acids (each corresponding to one of the above-mentioned genes) have been deposited. The kit can also include a plurality of labeled probes. Alternatively, the kit can include a plurality of polynucleotide sequences suitable as probes and a selection of labels suitable for customizing the included polynucleotide sequences, or other polynucleotide sequences at the discretion of the practitioner. Commonly, at least one included polynucleotide sequence corresponds to a control sequence, e.g., a "housekeeping" gene, β-actin or the like. Exemplary labels include, but are not limited to, a fluorophore, a dye, a radiolabel, an enzyme tag, that is linked to a nucleic acid primer.

In one embodiment, kits that are suitable for amplifying nucleic acid corresponding to the expressed RNA samples are provided. Such a kit includes reagents and primers suitable for use in any of the amplification methods described above. Alternatively, or additionally, the kits are suitable for amplifying a signal corresponding to hybridization between a probe and a target nucleic acid sample (e.g., deposited on a microarray).

In addition, one or more materials and/or reagents required for preparing a biological sample for gene expression analysis are optionally included in the kit. Furthermore, optionally included in the kits are one or more enzymes suitable for amplifying nucleic acids, including various polymerases (RT, Taq, etc.), one or more deoxynucleo tides, and buffers to provide the necessary reaction mixture for amplification.

Typically, the kits are employed for analyzing gene expression patterns using mRNA as the starting template. The mRNA template may be presented as either total cellular RNA or isolated mRNA; both types of sample yield comparable results. In other embodiments, the methods and kits described in the present invention allow quantitation of other products of gene expression, including tRNA, rRNA, or other transcription products.

Optionally, the kits of the invention further include software to expedite the generation, analysis and/or storage of data, and to facilitate access to databases. The software includes logical instructions, instructions sets, or suitable computer programs that can be used in the collection, storage and/or analysis of the data. Comparative and relational analysis of the data is possible using the software provided.

The kits optionally contain distinct containers for each individual reagent and/or enzyme component. Each component will generally be suitable as aliquoted in its respective container. The container of the kits optionally includes at least one vial, ampule, or test tube. Flasks, bottles and other container mechanisms into which the reagents can be placed and/or aliquoted are also possible. The individual containers of the kit are preferably maintained in close confinement for commercial sale. Suitable larger containers may include injection or blow-molded plastic containers into which the desired vials are retained. Instructions, such as written directions or videotaped demonstrations detailing the use of the kits of the present invention, are optionally provided with the kit.

In a further aspect, the present invention provides for the use of any composition or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.

A "subject" refers to a human and a non-human animal. Examples of a non-human animal include all vertebrates, e.g., mammals, such as non-human mammals, non-human primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat, and rabbit, and non-mammals, such as birds, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model.

A "test sample" or a "biological sample" as used herein may mean a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue isolated from animals. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, urine, effusions, amniotic fluid, ascitic fluid, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A biological sample may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods described herein in vivo. Archival tissues, such as those having treatment or outcome history, may also be used.

The term "inflammatory disorder" refers to a disorder that is characterized by abnormal or unwanted inflammation, such as sepsis, a systemic inflammatory response syndrome, and an autoimmune disease. Autoimmune diseases are disorders characterized by the chronic activation of immune cells under non- activating conditions. Examples include psoriasis, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, lupus, type I diabetes, primary biliary cirrhosis, and transplant. Other examples of inflammatory disorders where the methods of this invention can be used include asthma, myocardial infarction, stroke, inflammatory dermatoses (e.g., dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, dermatomyositis, and eosinophilic fasciitis), acute respiratory distress syndrome, fulminant hepatitis, hypersensitivity lung diseases (e.g., hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-type hypersensitivity, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, and ILD associated with rheumatoid arthritis), and allergic rhinitis. Additional examples also include myasthenia gravis, juvenile onset diabetes, glomerulonephritis, autoimmune throiditis, ankylosing spondylitis, systemic sclerosis, acute and chronic inflammatory diseases (e.g., systemic anaphylaxia or hypersensitivity responses, drug allergies, insect sting allergies, allograft rejection, and graft-versus-host disease), Sjogren's syndrome, thrombocytopenia (ITP), autoimmune hemolytic anemia (AHA), systemic lupus erythematosus (SLE), Kawsaki's disease (an acute vasculitic syndrome), sclerodema, rheumatoid arthritis (RA), chronic inflammatory demylinating polyneuropathy (CIDP), pemphigus and other conditions associated with autoantibody mediated inflammation.

The term "gene" used herein refers to a natural (e.g., genomic) or synthetic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5'- and 3 '-untranslated sequences). The coding region of a gene may be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA. A gene may also be a mRNA or cDNA corresponding to the coding regions (e.g., exons and miRNA) optionally comprising 5'- or 3 '-untranslated sequences linked thereto. A gene may also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3'-untranslated sequences linked thereto. The term also includes pseudogenes, which are dysfunctional relatives of known genes that have lost their protein- coding ability or are otherwise no longer expressed in a cell.

"Expression profile" as used herein refers to a genomic expression profile, e.g., an expression profile of mRNAs. Profiles may be generated by any convenient means for determining a level of a nucleic acid sequence e.g., quantitative hybridization of mRNA, cRNA, etc., quantitative PCR, ELISA for quantification, and the like, and allow the analysis of differential gene expression between two samples. A subject or patient sample, e.g., cells or a collection thereof, e.g., tissues, is assayed. Samples are collected by any convenient method, as known in the art. Nucleic acid sequences of interest are nucleic acid sequences that are found to be predictive, including the nucleic acid sequences of those described herein, where the expression profile may include expression data for 5, 10, 20, 25, 50, 100 or more of, including all of the listed nucleic acid sequences. The term "expression profile" may also mean measuring the abundance of the nucleic acid sequences in the measured samples.

"Differential expression" refers to qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue. Thus, a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus disease tissue. Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states. A qualitatively regulated gene will exhibit an expression pattern within a state or cell type that may be detectable by standard techniques. Some genes will be expressed in one state or cell type, but not in both. Alternatively, the difference in expression may be quantitative, e.g., in that expression is modulated, up-regulated, resulting in an increased amount of transcript, or down-regulated, resulting in a decreased amount of transcript. The degree to which expression differs need only be large enough to quantify via standard characterization techniques such as expression arrays, quantitative reverse transcriptase PCR, Northern analysis, and RNase protection.

"Nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein refers to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

The term "probe" as used herein refers to an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. A probe may be single stranded or partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. Probes may be directly labeled or indirectly labeled such as with biotin to which a streptavidin complex may later bind.

"Complement" or "complementary" as used herein to refer to a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. A full complement or fully complementary may mean 100% complementary base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

"Stringent hybridization conditions" as used herein refers to conditions under which a first nucleic acid sequence (e.g., probe) hybridizes to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence- dependent and be different in different circumstances, and can be suitably selected by one skilled in the art. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., about 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5xSSC, and 1% SDS, incubating at 42°C, or, 5xSSC, 1% SDS, incubating at 65°C, with wash in 0.2xSSC, and 0.1% SDS at 65°C. However, several factors other than temperature, such as salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to accomplish a similar stringency.

As used herein the term "reference value" refers to a value that statistically correlates to a particular outcome when compared to an assay result. In preferred embodiments, the reference value is determined from statistical analysis of studies that compare mRNA expression with known clinical outcomes. The reference value may be a threshold score value or a cutoff score value. Typically a reference value will be a threshold above (or below) which one outcome is more probable and below which an alternative threshold is more probable.

EXAMPLE 1

In this example, subjects and general materials and methods for carrying out various assays are disclosed.

Volunteer Subjects:

Healthy adult subjects were recruited by public advertisement and screened for inclusion in this study. Inclusion criteria for the study were normal general health as demonstrated by medical history and physical examination, complete blood count, and basic metabolic panel within normal lab limits. Exclusion criteria included a history of any acute or chronic disease, arrhythmia, recent history of alcohol, drug or medication ingestion, pregnancy or prior exposure to endotoxin in the experimental setting.

Upon accrual to the study, the subjects were admitted to the Clinical Research Center (CRC) at UMDNJ Robert Wood Johnson Medical School the afternoon prior to the study and a repeat examination confirmed that no changes in health status had occurred since enrollment. Female subjects underwent a urine pregnancy test. The subjects' characteristics are summarized in Table 1. The volunteer subjects were placed NPO at midnight prior to the endotoxin study day, and underwent intravenous fluid hydration (1 mL/kg-hr) until completion of the acute study phase.

Table 1. Volunteer subject and patient characteristics.

b LOS= length of stay

⁰ two patients received >5 units of RBC

Following admission, subjects were randomized to one of several study groups shown in Table 2 below.

Table 2

Subjects assigned to Groups B and D received a placebo infusion of physiologic saline prior to endotoxin administration. PBL samples obtained from these subjects prior to endotoxin infusion were used as baseline (Group A). Subjects assigned to Groups C and E received continuous intravenous infusion of Cortisol ^g/kg/min) for 12 hours starting 6 hours before endotoxin administration. Subjects assigned to Groups B-E received a one-time intravenous dose (2ng/kg) of endotoxin (NIH Clinical Center Reference Endotoxin; CC-RE- Lot2) at 0 hour (0900 clock time). Blood samples were drawn at 6 hours (Groups B and C) and 24 hours (Groups D and E) postendotoxin. Patients:

Patients were accrued from the adult Surgical ICU at Robert Wood Johnson University Hospital under a protocol approved by the Institutional Review Board of the Robert Wood Johnson Medical School.

The patient demographic characteristics are described in Table 1. An anticipated ICU stay of at least 72 hours and anticipated ultimate survival were utilized as inclusion criteria. Patients were excluded if they had a suspected or confirmed infection, received an organ transplant, required more than 6 units of blood transfusions and/or had severe traumatic brain injury (admitting GCS<8). Blood samples were first drawn within 1-5 days of ICU admission, and again 5-7 days later.

Blood samples were drawn in EDTA tubes, and centrifuged at 400 x g for 10 minutes. The plasma was removed, and the red blood cells/leukocytes pellet was treated with bicarbonate -buffered ammonium chloride lysing solution (0.1% potassium bicarbonate; 0.826% ammonium chloride in H20) at a ratio of 1 part red blood cell/ leukocytes to 20 parts lysing solution for 15 minutes in order to lyse the red blood cells. The leukocytes were then collected by centrifugation and washed once in lysing solution. After another centrifugation, a small aliquot of the leukocyte pellet was removed for performing a flow cytometric differential cell count on the healthy subjects. The leukocyte pellet was lysed in TRIzol™ solution (SIGMA, St. Louis MO), sheared 10 times with a 18 gauge needle, and frozen at -70°C.

Study Procedures:

Subjects were admitted to a clinical research center and managed as described in Alvarez, et al , J. Endotoxin Res 2007, 13(6):358-368 and Jan, et al, Ann Surg, 2009, 249(5) :750-756. Briefly, a standard dose of endotoxin (2 ng/kg, NIH Clinical Center Reference Endotoxin, CC-RE, Lot 2, Bethesda, Maryland) was administered intravenously over a five-minute period beginning at 09:00 AM or 09:00 PM (0 hour for the day- and night- subjects, respectively). The placebo subjects were administered saline (0.9% sodium chloride). All subjects had blood samples obtained at the indicated time-points relative to endotoxin or saline administration. Mean arterial pressure, heart rate, core temperature, and symptoms were recorded, and were consistent with those previously described. The peripheral intravenous catheter was removed at the completion of the study after each subject tolerated a regular diet. Preparation ofRNA, cDNA, and Labeled cRNA:

Total RNA:

Cell lysates in TRIzol™ were thawed and treated with chloroform. The RNA was isolated from the aqueous phase and precipitated with isopropyl alcohol. Following washing with alcohol, the RNA pellet was dried and dissolved in DEPC water. The quality and quantity of the isolated RNA was evaluated using the 2100 Bioanalyzer™ (AGILENT TECHNOLOGIES, Palo Alto, CA).

cDNA synthesis:

First strand cDNA synthesis was performed using reverse transcription (SUPERSCRIPTII, INVITROGEN, Carlsbad, CA) in a reaction containing 5μg of total RNA, T7-oligo (dt)24 primer, DTT, and dNTP mix. Second strand cDNA synthesis was then carried out by reaction of the first strand with DNA polymerase I, DNA ligase, and dNTP mix, followed by additional reaction with T4 DNA polymerase (INVITROGEN, Carlsbad, CA). Double-stranded cDNA was purified using the GeneChip Sample Cleanup Module (AFFYMETRIX, Santa Clara, CA).

cRNA Synthesis:

Biotinylated cRNA was synthesized from the double- stranded cDNA using GeneChip expression 3 '-amplification reagents for IVT labeling (AFFYMETRIX). This reaction uses MEGAscript T7 polymerase in the presence of a mixture of the four natural ribonucleotides and one biotin-conjugated analog. The biotinylated cRNA so-generated was then cleaned up using the GeneChip Sample Cleanup Module (AFFYMETRIX).

Microarray Analysis:

Steps outlined in this section were performed by the microarray core facility at UMDNJ. Following fragmentation of the biotinylated cRNA, 15μg was placed in hybridization cocktail, heated to 95° C, centrifuged and then hybridized to the Focus™ GeneChip microarray (AFFYMETRIX) for 16 hours at 45 °C. Chips were then washed, stained with streptavidin phycorerythrin and scanned on the Agilent Gene Array Scanner™ (AGILENT TECHNOLOGIES, Santa Clara, CA).

Analysis of Microarray Data:

Focus™ Gene chip data CEL files were imported, grouped and analyzed using

GeneSpring™ software (AGILENT TECHNOLOGIES, Santa Clara, CA). Primary analysis was carried out by log2 transformation followed by transformation to the median and RMA (quantile) normalization. Advanced significance analysis was performed on normalized- transformed data utilizing unpaired Student's t-tests. It was further defined significantly expressed probes as those with a P value < 0.05 and >1.2-fold change from baseline. Data were also exported for analysis by Ingenuity Pathway Analysis™ (INGENUITY, Palo Alto, CA) as previously described. The microarray data have been submitted to Gene Expression Omnibus (accession number pending).

qPCR:

Where indicated, RNA was extracted as described above and reversed transcribed to cDNA using High capacity cDNA Archive kit™ (APPLIED BIOSYSTEMS). Gene expression was analyzed in duplicate by quantitative real-time polymerase chain reaction (qPCR) using inventoried TaqMan® gene expression assays (APPLIED BIOSYSTEMS). A list of the gene expression assays can be found in Haimovich et al., Crit Care Med, 2010 38:751-758. The relative gene expression analysis was performed using the 2^"AACT method. The level of beta-2- microglobulin (B2M) expression was used as an internal reference.

Gene Expression Analyses in PBL:

Blood was drawn into Paxgene™ tubes (PREAN ALYTIX) . Total RNA was extracted as per PAXgene Blood RNA kit protocol (QIAGEN), quantified using AGILENT 2100 BIOANALYZER (AGILENT TECHNOLOGIES, Palo Alto, CA), and reversed transcribed to cDNA using High capacity cDNA Archive kit (Applied Biosystems). Gene expression was analyzed in duplicates by quantitative real-time (qRT) PCR using inventoried TaqMan® gene expression assay kits and the TaqMan universal PCR master mix (APPLIED BIOSYSTEMS). The relative gene expression analysis was performed using the 2^"AACT method. The level of beta-2 -micro globulin (B2M) expression was used as an internal reference, since the expression of B2M is not affected by endotoxin. Data are expressed as fold change relative to time 0. For the sample at time 0, AAC_T equals zero and 2° equals one, so that the fold change in gene expression relative to the control equals one, by definition.

Gene Expression Analyses in Leukocyte Cell Subpopulations:

For leukocytes isolation, blood was drawn into EDTA containing tubes (10ml) (BD

BIOSCIENCES). The EDTA containing tubes were centrifuged at 400 g for 10 minutes. The leukocyte pellets were mixed with erythrocytes lysis buffer (bicarbonate-buffered ammonium chloride solution, 0.826% NH₄C1, 0.1% KHC0₃, 0.0037% Na₄EDTA in H₂0) at a ratio of 20: 1

(lysis buffer: blood). The samples were incubated at room temperature until erythrocyte lysis was complete (~ 10 min). Leukocytes were recovered by centrifugation (400 g for 10 minutes at 4°C) and washed once with ice-cold phosphate-buffered saline. A small aliquot was removed for performing a flow cytometric differential cell count. The leukocytes were lysed in TRIZOL solution (INVITROGEN, Carlsbad, California), sheared, and frozen at -70° C. For monocytes isolation, blood was drawn directly into Vacutainer CPT™ tubes (BD BIOSCIENCES). RosetteSep™ human monocyte enrichment (STEM CELL TECHNOLOGIES, Vancouver, BC, Canada) cocktail (400 ul) was added to each tube, and the samples were incubated for 20 min at RT. The tubes were centrifuged at 1700 g for 25 minutes. The resulting interfacial layer was transferred to a new tube containing ice-cold PBS supplemented with 2% fetal bovine serum. The samples were next centrifuged at 500 g for 10 min. The resulting cell pellet was mixed with the erythrocyte lysis buffer for 10 min at RT. The monocytes were recovered by centrifugation (400 g for 10 min), and washed twice with PBS. A small aliquot was removed for performing a flow cytometric differential cell count. The remainder monocytes were lysed in TRIzol solution, sheared and frozen at -70° C. For neutrophil isolation, blood was drawn into EDTA containing tubes (10 ml) (BD BIOSCIENCES). Whole blood was diluted 1:2 with PBS, layered on Ficoll-Hypaque (SIGMA, St. Louis, MO) at a ratio of 2:1 (Ficolkblood), and centrifuged at 450 g for 30 minutes. The resulting red blood cells/neutrophil lower layer was mixed with an equal volume of 1.2% dextran (MW= 500,000) in saline and allowed to sediment at unit gravity for 30 minutes. The neutrophil enriched upper layer was transferred to another tube, and the monocytes were recovered by centrifugation (400 g for 10 min). The monocytes were than washed twice with PBS. A small aliquot was removed for performing a flow cytometric differential cell count. The remainder neutrophils were lysed in TRIzol solution, sheared and frozen at -70° C.

Cell purity was determined by 3 -colors flow cytometry. Cell samples were triple stained with CD66b-fluoroscein isothiocynate (FITC) (BECKMAN-COULTER, Miami, FL), CD2-phycoerythrin (PE) (BECTON-DICKENSON BIOSCIENCES, San Jose, CA) and CD33-peridinin-chlorophyll-protein complex, cyanin dye 5.5 PerCP-Cy5.5 (BD BIOSCIENCES) for 30 minutes at 4° C. After washing once with PBS containing 0.5% BSA, the cells were analyzed on a FACSCalibur ^ta flow cytometer (BD BIOSCIENCES). Granulocytes were identified as side-scatter high, FLl-high (CD66-FITC), T-cells as side scatter low, FL-2high (CD-2PE) and monocytes as side scatter-intermediate, FL3-high (CD33- PerCP-Cy5.5). Data were collected and analyzed using the CELLQuest^tm software. Only cell fractions >90% pure were used in subsequent studies. Cell lysates in TRIzol were processed according to the manufacturer instructions. The quality and quantity of the isolated RNA was evaluated using the 2100 Bioanalyzer (AGILENT TECHNOLOGIES, Palo Alto, CA). All subsequent steps were carried out as outlined above.

Cluster Analysis:

The mean of each individual gene expression levels was subtracted from the expression levels of each gene. The values were then cross-correlated across all genes within a subject. In the rare cases where a single time point was missing, it was imputed the missing value by averaging the time point before and after the missing one. The cross-correlation data were normalized to one, where one represents a perfect correlation between gene responses. The data were clustered into a tree form using the standard hierarchical Unweighted Pair Group Method with Arithmetic Mean algorithm (UPGMA), with one minus the cross-correlation coefficient as a measure of similarity. The distance between any two nodes is equivalent to the sum of the horizontal paths connecting the nodes. The heuristic algorithm iteratively joins the two nearest clusters (either individual nodes or groups of nodes) and defines the distances between any two nodes as equivalent to the sum of the horizontal paths connecting the nodes. All clustering operations were carried out in MatLab 2008a program.

TNFa and IL-6 Analyses:

Cytokine levels were determined by sandwich ELISA using monoclonal/polyclonal antibody pairs (R & D SYSTEMS, Minneapolis, MN) (additional information) and the vendor's protocol.

Steroid Hormones:

Hydrocortisone was determined by a direct radioimmunoassay based on a previous report. Cross reactivity of the antibody (additional information) was 5% with 11-deoxy- 170H-corticosterone and less than 0.5% with other relevant glucocorticoid and sex steroids.

Melatonin:

Plasma melatonin levels were determined using a direct Melatonin radioimmunoassay kit (LABOR DIAGNOSTIKA NORD GMBH & CO. KG; ROCKY MOUNTAINS DIAGNOSTICS, Colorado Springs, CO 08903) following the manufacturer protocol. The assay range was 0-1000 pg/ml.

Statistics:

Analysis of three groups or more was by one-way ANOVA with Newman-Keuls post- test. Two groups were compared by unpaired Student' s t test. The operations were carried out using Prism 4 software Version 4.0b (GRAPHPAD SOFTWARE, INC., La Jolla, CA). P values less than 0.05 were considered to be statistically significant.

EXAMPLE 2

This example describes results obtained from the differential gene expression study described above.

Array Data Analyses and Gene Selection:

To pursue this analysis, a database was compiled. The database included 38 Focus GeneChip® microarrays (AFFYMETRIX, Santa Clara CA) derived from studies of peripheral whole blood leukocytes (PBL) in seven human subjects study groups outlined in Table 2 (n>4 samples per group). PBL samples obtained from subjects administered saline were classified as Group A. This group served as a baseline for all analyses. PBL samples obtained from otherwise healthy subjects at 6- or 24-hours after an in vivo endotoxin challenge were classified in Groups B and D, respectively. Since it was unknown to what extent the post- injury pattern of gene expression was influenced by antecedent or co-existing neuroendocrine factors, assays were carried out to analyze gene expression patterns in PBL from subjects that were exposed to continuous intravenous infusion of Cortisol for 12 hours starting 6 hours before endotoxin (Groups C and E, respectively). In addition, the database included two matching PBL samples obtained from 5 patients. For 4 out of the 5 patients, the blood samples were obtained within 5 days (Group F) and 12 days of admission (Group G).

Differential Gene Expression In PBL Derived From In Vivo Endotoxin Challenged Subjects And Trauma Patients:

Prior studies indicated a maximal change in PBL gene expression at the 6 hour time point post endotoxin infusion in all volunteer subjects. Hence, this time-point was chosen to depict the influence of endotoxin. Expressed gene selection proceeded from the array database as outlined in FIG. 1. Arrays representing PBL obtained after an in vivo endotoxin challenge (Group B), or antecedent Cortisol plus endotoxin challenge (Group C), as well as those obtained from trauma patients within 5 days of admission (Group F), were independently compared to baseline. Gene probes that were significantly differentially expressed (both /?<0.05 and >1.2 fold-change) were then selected (FIG. 1A).

Out of the 8793 genes represented on the Focus GeneChip® microarrays, 2338 (27%) and 2962 (34%) genes were differentially expressed, by the criteria described above, in PBL 6 hours after challenge with endotoxin, without or with Cortisol, as compared to baseline (FIG. 1A). Of these, 1956 were common to PBL treated with endotoxin (Group B) and Cortisol plus endotoxin (Group C) (FIG. 1A).

Numerous genes (1581; 18%) were also differentially expressed (both /?<0.05 and >1.2 fold-change) in PBL obtained from trauma patients within the first 5 days of admission as compared to baseline values of normal subjects. Based on these similarities, 937 genes were significantly differentially expressed in all three groups (Group 1; FIGs. 1A).

Scatter plot analyses revealed that the gene expression trends were highly correlated among the three groups (FIG. IB). These data suggest a significant commonality among differentially expressed genes during the early, dynamic phase of TLR4-induced inflammation resulting from endotoxin infusion, and those differentially expressed in PBL in the early post- trauma time period.

Differential Gene Expression in PBL during Prolonged Injury:

Next, it was sought to determine which of the 937 genes that were differentially expressed during the peak of systemic inflammatory responses, and during the first several days after a trauma event, remain differentially expressed in PBL obtained at later time points of up to 12 days after ICU admission.

To that end, a set of 1136 genes that were differentially expressed in PBL obtained from trauma patients after 9-12 days of admission were selected (FIG. 1C). Then, assays were carried out to identify genes that were common to both this later injury phase group and those genes defined as Group 1 genes (FIG. ID). This resulted in the identification of 449 genes (5.4%) that persisted in differential expression in response to TLR4-induced systemic inflammation and/or injury. This group of TLR4 and injury responsive genes was referred to as "TIR" genes and listed in Table 3. This TIR gene group includes 273 genes that were down-regulated and 176 genes that were up-regulated ("D" and "U" in Table 3, respectively).

It was also sought to determine the expression trends of the 488 genes (= 937-449) that were differentially expressed in endotoxin challenged subjects and in trauma patients 0-5 days post admission, but which were no longer differentially expressed at later time points of up to 9-12 days after ICU admission. These genes were not included among those listed in Table 3. Of the 488 genes analyzed, 150 genes reversed their expression trends in all 4 subjects by 9-12 post ICU- admission. These 150 genes are listed in Table 4. Table 3

u 209413_at B4GALT2 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 2 u 212876_at B4GALT4 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 4

D 221234_s_at BACH2 BTB and CNC homology 1, basic leucine zipper transcription factor 2

U 205965_at BATF basic leucine zipper transcription factor, ATF- like

D 219528_s_at BCL11B B-cell CLL/lymphoma 11B (zinc finger

protein)

D 218285_s_at BDH2 3-hydroxybutyrate dehydrogenase, type 2

U 206956_at BGLAP bone gamma-carboxyglutamate (gla) protein

D 210538_s_at BIRC3 baculoviral IAP repeat-containing 3

D 202265_at BMI1 BMI1 polycomb ring finger oncogene

U 207595_s_at BMP1 bone morphogenetic protein 1

U 221454_at BOK BCL2 -related ovarian killer

U 205715_at BST1 bone marrow stromal cell antigen 1

D 211939_x_at BTF3 basic transcription factor 3

U 205690_s_at BUD31 BUD31 homolog (S. cerevisiae)

U 20578 l_at C16orf7 chromosome 16 open reading frame 7

D 211563_s_at C19orf2 chromosome 19 open reading frame 2

U 203052_at C2 complement component 2

U 217835_x_at C20orf24 chromosome 20 open reading frame 24

U 206656_s_at C20orf3 chromosome 20 open reading frame 3

D 221158_at C21orf66 chromosome 21 open reading frame 66

U 204968_at C6orf47 chromosome 6 open reading frame 47

D 20930 l_at CA2 carbonic anhydrase II

D 20481 l_s_at CACNA2D2 calcium channel, voltage-dependent, alpha

2/delta subunit 2

D 219714_s_at CACNA2D3 calcium channel, voltage-dependent, alpha

2/delta subunit 3

U 210244_at CAMP cathelicidin antimicrobial peptide

D 203356_at CAPN7 calpain 7

D 211208_s_at CASK calcium/calmodulin-dependent serine protein kinase (MAGUK family)

D 22220 l_s_at CASP8AP2 caspase 8 associated protein 2

D 209682_at CBLB Cas-Br-M (murine) ecotropic retroviral

transforming sequence b

U 212816_s_at CBS cystathionine-beta-synthase

D 200953_s_at CCND2 cyclin D2

U 204826_at CCNF cyclin F

D 208304_at CCR3 chemokine (C-C motif) receptor 3

D 206337_at CCR7 chemokine (C-C motif) receptor 7

U 219669_at CD 177 CD 177 molecule

D 20583 l_at CD2 CD2 molecule

D 207315_at CD226 CD226 molecule

D 206545_at CD28 CD28 molecule

D 206804_at CD3G CD3g molecule, gamma (CD3-TCR complex) u 200663_at CD63 CD63 molecule

D 209795_at CD69 CD69 molecule

D 202717_s_at CDC 16 cell division cycle 16 homolog (S. cerevisiae)

D 207318_s_at CDC2L5 cell division cycle 2-like 5 (cholinesterase- related cell division controller)

U 206824_at CES4 carboxylesterase 4-like

U 203953_s_at CLDN3 claudin 3

D 208925_at CLDND1 claudin domain containing 1

D 220132_s_at CLEC2D C-type lectin domain family 2, member D

D 218250_s_at CNOT7 CCR4-NOT transcription complex, subunit 7

D 218142_s_at CRBN cereblon

D 202979_s_at CREBZF CREB/ATF bZIP transcription factor

U 20753 l_at CRYGC crystallin, gamma C

D 219939_s_at CSDE1 cold shock domain containing El, RNA- binding

U 210140_at CST7 cystatin F (leukocystatin)

D 203758_at CTSO cathepsin O

U 210816_s_at CYB561 cytochrome b-561

U 200932_s_at DCTN2 dynactin 2 (p50)

U 214909_s_at DDAH2 dimethylarginine dimethylaminohydrolase 2

D 208895_s_at DDX18 DEAD (Asp-Glu-Ala-Asp) box polypeptide

18

D 201386_s_at DHX15 DEAH (Asp-Glu-Ala-His) box polypeptide

15

D 213598_at DIMT1L DIM1 dimethyladenosing transef erase 1-like

U 204008_at DNAL4 dynein, axonemal, light chain 4

U 207192_at DNASE1L2 deoxyribonuclease I-like 2

D 201697_s_at DNMT1 DNA (cytosine-5-)-methyltransferase 1

D 204794_at DUSP2 dual specificity phosphatase 2

D 202969_at DYRK2 dual- specificity tyrosine-(Y)-phosphorylation regulated kinase 2

U 218660_at DYSF dysferlin, limb girdle muscular dystrophy 2B

(autosomal recessive)

U 201749_at ECE1 endothelin converting enzyme 1

D 206559_x_at EEF1A1 eukaryotic translation elongation factor 1 alpha 1

U 218825_at EGFL7 EGF-like-domain, multiple 7

D 208697_s_at EIF3E eukaryotic translation initiation factor 3, subunit E

D 200912_s_at EIF4A2 eukaryotic translation initiation factor 4A, isoform 2

U 206338_at ELAVL3 ELAV (embryonic lethal, abnormal vision,

Drosophila)-like 3 (Hu antigen C)

D 204143_s_at ENOSF1 enolase superfamily member 1

U 202894_at EPHB4 EPH receptor B4

D 20754 l_s_at EXOSC10 exosome component 10

U 201995_at EXT1 exostoses (multiple) 1

U 204714_s_at F5 coagulation factor V (proaccelerin, labile factor) u 202535_at FADD Fas (TNFRSF6)-associated via death domain

D 221601_s_at FAIM3 Fas apoptotic inhibitory molecule 3

D 206848_at FAM36A /// family with sequence similarity 36, member

HOXA7 A /// homeobox A7

U 204232_at FCER1G Fc fragment of IgE, high affinity I, receptor for; gamma polypeptide

U 216950_s_at FCGR1A /// Fc fragment of IgG, high affinity la, receptor

FCGR (CD64)

U 221385_s_at FFAR3 free fatty acid receptor 3

D 210607_at FLT3LG fms-related tyrosine kinase 3 ligand

U 20637 l_at FOLR3 folate receptor 3 (gamma)

D 204299_at FUSIP1 FUS interacting protein (serine/arginine-rich)

1

D 210105_s_at FYN F YN oncogene related to SRC, FGR, YES

U 207574_s_at GADD45B growth arrest and DNA-damage-inducible, beta

U 210446_at GATA1 GATA binding protein 1 (globin transcription factor 1)

D 209604_s_at GATA3 GATA binding protein 3

D 202270_at GBP1 guanylate binding protein 1, interferon- inducible, 67kDa

U 221360_s_at GHSR growth hormone secretagogue receptor

D 207153_s_at GLMN glomulin, FKBP associated protein

U 35820_at GM2A GM2 ganglioside activator

D 218458_at GMCL1 germ cell-less homolog 1 (Drosophila)

U 205349_at GNA15 guanine nucleotide binding protein (G

protein), alpha 15 (Gq class)

D 20065 l_at GNB2L1 guanine nucleotide binding protein (G

protein), beta polypeptide 2-like 1

D 37145_at ONLY granulysin

U 208519_x_at GNRH2 gonadotropin-releasing hormone 2

U 210640_s_at GPER G protein-coupled estrogen receptor 1

D 205419_at GPR183 G protein-coupled receptor 183

D 212070_at GPR56 G protein-coupled receptor 56

U 209409_at GRB10 growth factor receptor-bound protein 10

D 218343_s_at GTF3C3 general transcription factor IIIC, polypeptide

3, 102kDa

U 211275_s_at GYG1 glycogenin 1

D 205488_at GZMA granzyme A (granzyme 1 , cytotoxic T- lymphocyte-associated serine esterase 3)

D 210164_at GZMB granzyme B (granzyme 2, cytotoxic T- lymphocyte-associated serine esterase 1)

D 206666_at GZMK granzyme K (granzyme 3 ; tryptase II)

U 200989_at HIF1A hypoxia inducible factor 1 , alpha subunit

(basic helix-loop-helix transcription factor)

U 208569_at HIST1H2AB histone cluster 1 , H2ab

U 214522_x_at HIST1H2AD histone cluster 1, H2ad /// histone cluster 1,

/// HIS H3d u 214472_at HIST1H2AD histone cluster 1, H2ad /// histone cluster 1,

/// HIS H3d

u 214634_at HIST1H4B Histone cluster 1, H4b

u 208076_at HIST1H4D histone cluster 1 , H4d

u 20855 l_at HIST1H4G histone cluster 1 , H4g

u 205936_s_at HK3 hexokinase 3 (white cell)

D 202983_at HLTF helicase-like transcription factor

U 214438_at HLX H2.0-like homeobox

D 218152_at HMG20A high-mobility group 20A

D 209786_at HMGN4 high mobility group nucleosomal binding domain 4

U 217755_at HN1 hematological and neurological expressed 1

D 200016_x_at HNRNPA1 heterogeneous nuclear ribonucleoprotein Al

D 221919_at HNRNPA1 heterogeneous nuclear ribonucleoprotein Al

/// LOC7 /// hypothetical LOC728844

D 20103 l_s_at HNRNPH1 heterogeneous nuclear ribonucleoprotein HI

(H)

D 201406_at HNRNPH2 heterogeneous nuclear ribonucleoprotein H2

/// RPL3 (H) /// ribosomal protein L36a ///

D 208766_s_at HNRNPR heterogeneous nuclear ribonucleoprotein R

D 209067_s_at HNRPDL heterogeneous nuclear ribonucleoprotein D- like

D 204544_at HPS 5 Hermansky-Pudlak syndrome 5

D 219212_at HSPA14 heat shock 70kDa protein 14

D 201565_s_at ID2 inhibitor of DNA binding 2, dominant

negative helix-loop-helix protein

U 201631_s_at IER3 immediate early response 3

U 215712_s_at IGFALS insulin-like growth factor binding protein, acid labile subunit

D 204773_at IL11RA interleukin 11 receptor, alpha

U 207844_at IL13 interleukin 13

U 212657_s_at IL1RN interleukin 1 receptor antagonist

D 20529 l_at IL2RB interleukin 2 receptor, beta

D 205798_at IL7R interleukin 7 receptor

U 204169_at IMPDH1 IMP (inosine monophosphate) dehydrogenase

1

U 205258_at INHBB inhibin, beta B

D 213416_at ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)

U 21633 l_at ITGA7 integrin, alpha 7

D 211339_s_at ITK IL2-inducible T-cell kinase

U 206842_at KCND1 potassium voltage-gated channel, Shal-related subfamily, member 1

U 207635_s_at KCNH1 potassium voltage-gated channel, subfamily H

(eag-related), member 1

U 20966 l_at KIFC3 kinesin family member C3

D 211410_x_at KIR2DL5A killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 5 A D 211532_x_at KIR2DS1 /// killer cell immunoglobulin-like receptor, two

KIR2D domains, short cytoplasmic tail, 1 /tail, 2 / tail, 4

D 208203_x_at KIR2DS5 killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 5

D 207314_x_at KIR3DL2 /// killer cell immunoglobulin-like receptor, three

LOC72 domains, long cytoplasmic tail, 2 ///

D 217906_at KLHDC2 kelch domain containing 2

D 221221_s_at KLHL3 kelch-like 3 (Drosophila)

D 214470_at KLRB1 killer cell lectin- like receptor subfamily B, member 1

D 206785_s_at KLRC1 killer cell lectin-like receptor subfamily C, member 1 /// member 2

D 207723_s_at KLRC3 killer cell lectin-like receptor subfamily C, member 3

D 207795_s_at KLRD1 killer cell lectin-like receptor subfamily D, member 1

D 220646_s_at KLRF1 killer cell lectin-like receptor subfamily F, member 1

D 210288_at KLRG1 killer cell lectin-like receptor subfamily G, member 1

D 205821_at KLRK1 killer cell lectin-like receptor subfamily K, member 1

D 202020_s_at LANCL1 LanC lantibiotic synthetase component C-like

1 (bacterial)

D 20489 l_s_at LCK lymphocyte- specific protein tyrosine kinase

D 201030_x_at LDHB lactate dehydrogenase B

D 221558_s_at LEF1 lymphoid enhancer-binding factor 1

U 206230_at LHX1 LIM homeobox 1

D 214035_x_at LOC399491 GPS, PLAT and transmembrane domain- containing protein

D 216902_s_at LOC653390// RNA polymerase I transcription factor

/LOCRRN3 homolog (S. cerevisiae) pseudogene ///

U 20438 l_at LRP3 low density lipoprotein receptor-related

protein 3

U 210128_s_at LTB4R leukotriene B4 receptor

U 220130_x_at LTB4R2 leukotriene B4 receptor 2

U 204970_s_at MAFG v-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian)

U 20657 l_s_at MAP4K4 mitogen- activated protein kinase kinase

kinase kinase 4

U 205050_s_at MAPK8IP2 mitogen- activated protein kinase 8 interacting protein 2

U 205819_at MARCO macrophage receptor with collagenous

structure

D 200626_s_at MATR3 matrin 3

D 218440_at MCCC1 methylcrotonoyl- Coenzyme A carboxylase 1

(alpha)

D 203497_at MED1 mediator complex subunit 1

D 203939_at NT5E 5 '-nucleotidase, ecto (CD73)

U 219708_at NT5M 5',3'-nucleotidase, mitochondrial

D 217802_s_at NUCKS1 nuclear casein kinase and cyclin-dependent kinase substrate 1

U 202155_s_at NUP214 nucleoporin 214kDa

D 202073_at OPTN optineurin

U 219475_at OSGIN1 oxidative stress induced growth inhibitor 1

D 208717_at OXA1L oxidase (cytochrome c) assembly 1-like

D 214615_at P2RY10 purinergic receptor P2Y, G-protein coupled,

10

D 215157_x_at PABPC1 poly(A) binding protein, cytoplasmic 1

D 208113_x_at PABPC3 poly(A) binding protein, cytoplasmic 3

D 202760_s_at PALM2- PALM2-AKAP2 readthrough transcript

AKAP2

U 211867_s_at PCDHA10 protocadherin alpha 10

D 202174_s_at PCM1 pericentriolar material 1

D 203660_s_at PCNT pericentrin

U 207634_at PDCD1 programmed cell death 1

U 206444_at PDE1B phosphodiesterase IB, calmodulin-dependent

U 221957_at PDK3 pyruvate dehydrogenase kinase, isozyme 3

U 202464_s_at PFKFB3 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3

U 207384_at PGLYRP1 peptidoglycan recognition protein 1

U 20849 l_s_at PGM5 phosphoglucomutase 5

U 204746_s_at PICKl protein interacting with PRKCA 1

D 221689_s_at PIGP phosphatidylinositol glycan anchor

biosynthesis, class P

U 208277_at PITX3 paired-like homeodomain 3

D 219024_at PLEKHA1 pleckstrin homology domain containing, family A (phosphoinositide binding specific)

D 219700_at PLXDC1 plexin domain containing 1

U 220923_s_at PNMA3 paraneoplastic antigen MA3

D 203622_s_at PNOl partner of NOB 1 homolog (S. cerevisiae)

D 20581 l_at POLG2 polymerase (DNA directed), gamma 2, accessory subunit

D 202466_at POLS polymerase (DNA directed) sigma

D 201293_x_at PPIA peptidylprolyl isomerase A (cyclophilin A)

D 200726_at PPP1CC protein phosphatase 1 , catalytic subunit, gamma isoform

D 202165_at PPP1R2 protein phosphatase 1 , regulatory (inhibitor) subunit 2

D 207830_s_at PPP1R8 protein phosphatase 1 , regulatory (inhibitor) subunit 8

D 203338_at PPP2R5E protein phosphatase 2, regulatory subunit B', epsilon isoform

U 220654_at PPY2 pancreatic polypeptide 2

D 214617_at PRF1 perforin 1 (pore forming protein)

D 202742_s_at PRKACB protein kinase, cAMP-dependent, catalytic, beta D 209678_s_at PRKCI protein kinase C, iota

D 210039_s_at PRKCQ protein kinase C, theta

U 221443_x_at PRLH prolactin releasing hormone

U 20729 l_at PRRG4 proline rich Gla (G-carboxyglutamic acid) 4

(transmembrane)

D 202458_at PRSS23 protease, serine, 23

D 205961_s_at PSIP1 PC4 and SFRS 1 interacting protein

D 1209337_at PSIP1 PC4 and SFRS 1 interacting protein 1

D 218967_s_at PTER phosphodiesterase related

D 200627_at PTGES3 prostaglandin E synthase 3 (cytosolic)

D 205171_at PTPN4 protein tyrosine phosphatase, non-receptor type 4 (megakaryocyte)

D 204020_at PURA purine -rich element binding protein A

U 203149_at PVRL2 poliovirus receptor-related 2 (herpesvirus entry mediator B)

D 201606_s_at PWP1 PWP1 homolog (S. cerevisiae)

U 201482_at QSOX1 quiescin Q6 sulfhydryl oxidase 1

U 202252_at RAB13 RAB13, member RAS oncogene family

U 204214_s_at RAB32 RAB32, member RAS oncogene family

D 219151_s_at RABL2A /// RAB, member of RAS oncogene family-like

RABL2 2A /// 2B

U 206103_at RAC3 ras-related C3 botulinum toxin substrate 3

(rho family, small GTP binding protein Rac3)

D 221830_at RAP2A RAP2A, member of RAS oncogene family

U 203749_s_at RARA retinoic acid receptor, alpha

D 212706_at RASA4 RAS p21 protein activator 4 pseudogene ///

RAS p21 protein activator 4

D 205590_at RASGRP1 RAS guanyl releasing protein 1 (calcium and

DAG-regulated)

D 208319_s_at RBM3 RNA binding motif (RNP1, RRM) protein 3

D 201967_at RBM6 RNA binding motif protein 6

U 213520_at RECQL4 RecQ protein-like 4

U 205645_at REPS2 RALBPl associated Eps domain containing 2

U 220570_at RETN resistin

U 203823_at RGS3 regulator of G-protein signaling 3

U 20521 l_s_at RIN1 Ras and Rab interactor 1

U 201785_at RNASE1 ribonuclease, RNase A family, 1 (pancreatic)

U 219104_at RNF141 ring finger protein 141

D 202683_s_at RNMT RNA (guanine-7-) methyltransferase

U 205806_at ROM1 retinal outer segment membrane protein 1

D 210479_s_at RORA RAR-related orphan receptor A

D 201528_at RPA1 replication protein Al, 70kDa

D 200036_s_at RPL10A ribosomal protein LlOa

D 200010_at RPL11 ribosomal protein LI 1

D 200074_s_at RPL14 ribosomal protein LI 4 /// ribosomal protein

L14 pseudogene 1

D 221476_s_at RPL15 ribosomal protein LI 5

D 200038_s_at RPL17 ribosomal protein LI 7 D 200029_at RPL19 ribosomal protein LI 9

D 200012_x_at RPL21 ribosomal protein L21

D 208768_x_at RPL22 ribosomal protein L22

D 200888_s_at RPL23 ribosomal protein L23

D 208825_x_at RPL23A ribosomal protein L23a

D 200013_at RPL24 ribosomal protein L24

D 203034_s_at RPL27A ribosomal protein L27a

D 200062_s_at RPL30 ribosomal protein L30

D 200026_at RPL34 ribosomal protein L34

D 200002_at RPL35 ribosomal protein L35

D 200092_s_at RPL37 ribosomal protein L37

D 208695_s_at RPL39 ribosomal protein L39

D 200089_s_at RPL4 ribosomal protein L4

D 200937_s_at RPL5 ribosomal protein L5

D 200034_s_at RPL6 ribosomal protein L6

D 200717_x_at RPL7 ribosomal protein L7

D 200032_s_at RPL9 ribosomal protein L9

D 200909_s_at RPLP2 ribosomal protein, large, P2

D 213377_x_at RPS12 ribosomal protein S12

D 200018_at RPS13 ribosomal protein S13

D 20078 l_s_at RPS15A ribosomal protein SI 5a

D 201049_s_at RPS18 ribosomal protein S18

D 200834_s_at RPS21 ribosomal protein S21

D 200926_at RPS23 ribosomal protein S23

D 20006 l_s_at RPS24 ribosomal protein S24

D 20009 l_s_at RPS25 ribosomal protein S25

D 20074 l_s_at RPS27 ribosomal protein S27

D 200017_at RPS27A ribosomal protein S27a

D 201094_at RPS29 ribosomal protein S29

D 200099_s_at RPS3A ribosomal protein S3A

D 200933_x_at RPS4X ribosomal protein S4, X-linked

D 20008 l_s_at RPS6 ribosomal protein S6

U 203379_at RPS6KA1 ribosomal protein S6 kinase, 90kDa,

polypeptide 1

D 200082_s_at RPS7 /// ribosomal protein S7 /// ribosomal protein S7

RPS7P11 pseudogene 11

D 200858_s_at RPS8 ribosomal protein S8

D 217915_s_at RSL24D1 ribosomal L24 domain containing 1

U 34408_at RTN2 reticulon 2

D 204197_s_at RUNX3 runt-related transcription factor 3

U 205863_at S100A12 S100 calcium binding protein Al

D 2204642_at S1PR1 sphingosine- 1 -phosphate receptor 1

U 20005 l_at SART1 squamous cell carcinoma antigen recognized by T cells

D 203408_s_at SATB1 SATB homeobox 1

U 210364_at SCN2B sodium channel, voltage-gated, type II, beta

U 20524 l_at SC02 SCO cytochrome oxidase deficient homolog 2

(yeast) D 201339_s_at SCP2 sterol carrier protein 2

D 202542_s_at SCYE1 small inducible cytokine subfamily E,

member 1 (endothelial monocyte- activating)

U 203090_at SDF2 stromal cell-derived factor 2

U 220778_x_at SEMA6B sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6B

U 211429_s_at SERPINA1 serpin peptidase inhibitor, clade A (alpha- 1 antiproteinase, antitrypsin), member 1

D 218346_s_at SESN1 sestrin 1

D 200686_s_at SFRS11 splicing factor, arginine/serine-rich 11

D 221268_s_at SGPP1 sphingosine- 1 -phosphate phosphatase 1

D 210116_at SH2D1A SH2 domain protein 1A

U 202896_s_at SIRPA signal-regulatory protein alpha

D 207974_s_at SKP1 S-phase kinase-associated protein 1

U 210423_s_at SLC11A1 solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1

U 207567_at SLC13A2 solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 2

U 211576_s_at SLC19A1 solute carrier family 19 (folate transporter), member 1

D 205097_at SLC26A2 solute carrier family 26 (sulfate transporter), member 2

U 202499_s_at SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3

D 218237_s_at SLC38A1 solute carrier family 38, member 1

D 213164_at SLC5A3 solute carrier family 5 (sodium/myo-inositol cotransporter), member 3

U 202219_at SLC6A8 solute carrier family 6 (neurotransmitter transporter, creatine), member 8

D 203579_s_at SLC7A6 solute carrier family 7 (cationic amino acid transporter, y+ system), member 6

U 20302 l_at SLPI secretory leukocyte peptidase inhibitor

D 211988_at SMARCE1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin

D 203852_s_at SMN1 /// survival of motor neuron 1 , telomeric ///

SMN2 survival of motor neuron 2, centromeric

U 205300_s_at SNRNP35 small nuclear ribonucleoprotein 35kDa

(U11/U12)

U 219257_s_at SPHK1 sphingosine kinase 1

D 218638_s_at SP0N2 spondin 2, extracellular matrix protein

U 217995_at SQRDL sulfide quinone reductase-like (yeast)

D 201273_s_at SRP9 signal recognition particle 9kDa

D 201225_s_at SRRM1 serine/arginine repetitive matrix 1

D 201138_s_at SSB Sjogren syndrome antigen B (autoantigen La)

D 208666_s_at ST13 suppression of tumorigenicity 13 (colon carcinoma) (Hsp70 interacting protein)

U 205346_at ST3GAL2 ST3 beta-galactoside alpha-2,3- sialyltransferase 2 u 203759_at ST3GAL4 ST3 beta-galactoside alpha-2,3- sialyltransferase 4

D 220059_at STAP1 signal transducing adaptor family member 1

D AFFX- STAT1 signal transducer and activator of

HUMISG transcription 1, 91kDa

D 206118_at STAT4 signal transducer and activator of

transcription 4

U 20760 l_at SULT1B1 sulfotransferase family, cytosolic, IB,

member 1

D 217833_at SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting protein

U 208048_at TACR1 tachykinin receptor 1

D 202840_at TAF15 TAF15 RNA polymerase II, TATA box

binding protein (TBP)-associated factor, 68kDa

U 204986_s_at TAOK2 TAO kinase 2

D 202813_at T ARB PI TAR (HIV-1) RNA binding protein 1

U 220634_at TBX4 T-box 4

D 202396_at TCERG1 transcription elongation regulator 1

D 203753_at TCF4 transcription factor 4

D 203449_s_at TERF1 telomeric repeat binding factor (NIMA- interacting) 1

D 20473 l_at TGFBR3 transforming growth factor, beta receptor III

D 204064_at THOC1 THO complex 1

U 209418_s_at THOC5 THO complex 5

U 217847_s_at THRAP3 thyroid hormone receptor associated protein 3

D 219477_s_at THSD1 /// thrombospondin, type I, domain containing 1

THSD1P /// pseudogene

U 203167_at TIMP2 TIMP metallopeptidase inhibitor 2

U 203437_at TMEM11 transmembrane protein 11

D 200847_s_at TMEM66 transmembrane protein 66

D 210260_s_at TNFAIP8 tumor necrosis factor, alpha-induced protein 8

U 202807_s_at TOM1 target of mybl (chicken)

D 200662_s_at TOMM20 translocase of outer mitochondrial membrane

20 homolog (yeast)

U 20342 l_at TP53I11 tumor protein p53 inducible protein 11

U 210609_s_at TP53I3 tumor protein p53 inducible protein 3

U 216485_s_at TPSAB1 tryptase alpha/beta 1

D 205599_at TRAF1 TNF receptor-associated factor 1

D 204352_at TRAF5 TNF receptor-associated factor 5

U 35254_at TRAFD1 TRAF-type zinc finger domain containing 1

D 208662_s_at TTC3 tetratricopeptide repeat domain 3

D 206828_at TXK TXK tyrosine kinase

U 21801 l_at UBL5 ubiquitin-like 5

U 203234_at UPP1 uridine phosphorylase

D 1218386_x_a USP16 ubiquitin specific peptidase 16

t

D 206624_at USP9Y ubiquitin specific peptidase 9, Y-linked D 209486_at UTP3 UTP3, small subunit (SSU) processome component, homolog (S. cerevisiae)

D 218715_at UTP6 UTP6, small subunit (SSU) processome component, homolog (yeast)

U 208780_x_at VAPA VAMP (vesicle-associated membrane

protein)-associated protein A, 33kDa

U 204022_at WWP2 WW domain containing E3 ubiquitin protein ligase 2

U 213081_at ZBTB22 zinc finger and BTB domain containing 22

U 218078_s_at ZDHHC3 zinc finger, DHHC-type containing 3

D 201368_at ZFP36L2 zinc finger protein 36, C3H type-like 2

D 202136_at ZMYND11 zinc finger, MYND domain containing 11

D 219571_s_at ZNF12 zinc finger protein 12

D 20693 l_at ZNF141 zinc finger protein 141

U 206416_at ZNF205 zinc finger protein 205

D 203707_at ZNF263 zinc finger protein 263

D 219228_at ZNF331 zinc finger protein 331

D 206059_at ZNF91 zinc finger protein 91

Table 4

209037_s_at EHD1 202789_at PLCG1 218082_s_at UBP1

20593 l_s_at CREB5 219041_s_at REPIN1 218316_at TIMM9

201725_at CDC123 202109_at ARFIP2 209657_s_at HSF2

214400_at INSL3 205019_s_at VIPR1 210017_at MALT1

206513_at AIM2 208904_s_at RPS28 203804_s_at CROP

201222_s_at RAD23B 206492_at FHIT 205571_at LIPT1

214697_s_at ROD1 201272_at AKR1B 1 203685_at BCL2

220005_at P2RY13 200086_s_at COX4I1 207655_s_at BLNK

205896_at SLC22A4 203664_s_at POLR2D 214567_s_at XCLl ///

XCL2

201642_at IFNGR2 209014_at MAGED1 207840_at CD160

The TIR genes selected as outlined in FIG. 1, plus those from the 24 hours post- endotoxin groups (Table 2; Groups D and E) were subjected to hierarchical cluster analysis. The clustering analysis defined two dominant groups. Cluster 1 included both baseline samples and all PBL samples derived from subjects at 24 hours after endotoxin. Cluster 2 included all the PBL samples derived from subjects at 6 hours post-endotoxin challenge as well as the trauma patient samples.

Assays were carried out to also examine the TIR gene expression trends using a published database (GEO GSE3284) that includes microarray data derived from 4 previously reported endotoxin challenged subjects at 0, 2 4, 6, 9 and 24 hours post challenge, and 4 control subjects studied at parallel time points. The TIR genes showed a robust response in all endotoxin-challenged subjects, and a return to baseline by 24 hours post treatment. Furthermore, hierarchical cluster analysis revealed two dominant clusters. Cluster 1 included a total of 30 samples representing 26 control samples plus 4 PBL samples obtained from endotoxin challenged subjects at 24 hours post-infusion. Cluster 2 included all the PBL samples obtained between 2 and 9 hours post-infusion. This significant degree of correspondence between a prior endotoxin challenged population and the present volunteers group confirms the fidelity of the above baseline and endotoxin challenged-subjects analysis.

TIR Genes Pathways and Interactions:

The TIR genes group includes 273 down-regulated and 176 up-regulated genes (Table

3). This group of differentially expressed genes includes an abundance of RPL (ribosomal proteins associated with large 60S ribosomal subunit) and RPS genes (ribosomal proteins associated with small 40S ribosomal subunit) genes. Furthermore, 50 of the 53 RPL/KPS genes (encoding ribosomal proteins L or S) were down-regulated. Among the down-regulated TIR genes were also 3 EIF/EEF genes (EIF3E, EIF4A2, and EEF1A1), which encode translation initiation factors, and 6 HNRNP genes (HNRNPA1, HNRNPA1///LOC7, HNRNPH1, HNRNPH2///RPL3, HNRNPR, and HNRPDL), which regulate premRNA processing and other aspects related to mRNA metabolism.

The expression data were analyzed through the use of Ingenuity Pathway Analysis (Ingenuity® systems, www.ingenuity.com) as previously described. This analysis classified the TIR genes into 5 main modules, each representing 140 genes (the maximum number of genes that the program associates with each module). Three out of the top 5 modules, which include approximately 230 TIR genes in total, are related to protein synthesis pathways. Two additional pathways, a lipid metabolism pathway, and a cellular assembly and organization pathway, included, respectively, 71- and 68-TIR gene matches.

The top matching module shown in FIG. 3 includes 99 TIR genes. Myc, a global transcription regulator of many cellular processes, including ribosomal biogenesis and protein synthesis, is the focal point for the most densely populated node encompassing numerous RPL/RPS genes. This large number of suggested interactions is not surprising given that more that 600 genes, including 48 transcription factors, were identified as direct Mvoregulated gene targets in human B lymphoid tumor cells alone. Furthermore, TIDBase, a web-based public resource supported by the type 1 diabetes (TID) research community (www.tldbase.org), identified more than 1400 Myorelated interactions. The implied reduction of PBL protein synthesis capacity is highly significant. A decline in transcripts associated with transcription was first observed in PBL obtained from endotoxin-challenged subjects.

However, the endotoxin-induced changes in PBL gene expression were all transient, with recovery within 24 hours. By contrast, the identification of a similar and persistent gene expression signature in PBL obtained from trauma patients 1-12 days post-admission indicates that the translational function of circulating leukocytes is consistently reprogrammed to a lower state.

Among the up-regulated TIR genes were several genes that are known to be associated with glycolysis. These include PFKFB3, encoding 6-phosphofructo-2-kinase (PFK-2), and HK3, encoding hexokinase 3. PFK-2 is a bifunctional enzyme that catalyzes the synthesis and degradation of fructose 2,6- biphosphate, which in turn, stimulates 6-phosphofructo-l -kinase, the key regulator of mammalian glycolysis. An increase in PFKFB3 (also known as iPFK2) expression has been documented in endotoxin- treated cultured human monocytes. Hexokinase 3 phosphorylates glucose to produce glucose-6-phosphate, the first intermediate in glycolysis. Also observed was an upregulation of SLC2A3, encoding the glucose transporter Glut 3, and PDK3, encoding pyruvate dehydrogenase kinase (PDK). PDK is an inhibitor of pyruvate dehydrogense complex, which is positioned at the junction between glycolysis and the TCA cycle. In cancer cells, an increase in PDK3 expression was associated with an increase in lactic acid production, which is indicative of a decrease in mitochondrial respiration. These collective changes in gene expression predict an increase in glucose consumption and glycolysis. This is supported by studies in endotoxin-challenged rats, wherein an increase in glucose utilization in multiple organs was observed within hours of an endotoxin or TNFa challenge. These data indicate that the systemic conditions induced by acute TLR4 ligation, resulting in enhanced PBL glycolysis, also persist for an extended period after trauma.

The above-mentioned glycolysis genes, RPL/KPS genes, EIF/EEF, and HNRNP genes, which are listed in Table 3 or 4, can be used to practice the methods of this invention.

EXAMPLE 3

Included among the suppressed TIR genes was also Rora, one of the key regulators of the circadian clock. In this example, assays were carried out to determine the expression status of a number of other key regulators of the circadian clock, including Clock, Cryl, Cry2, Per3, and Rora, in a subset of these surgical ICU patient samples.

The circadian clock is an autoregulatory feedback network of transcription factors and proteins whose activity and/or availability cycle with a periodicity of approximately 24 h. The central "master" clock controlling behavioral circadian rhythms is located in the suprachiasmatic nucleus (SCN) within the brain hypothalamus. The central clock both regulates and receives inputs from peripheral clocks present in most tissues, including PBL. Multiple circadian clock genes, including Clock, Cryl, Cry2, Per3, and Rora, are significantly suppressed within 2 hours after an endotoxin challenge and remain suppressed for up to 17 hours post-infusion. Assays were carried out to determine the status of Clock, Cryl, Cry2, Per3, and Rora expression in a subset of these surgical ICU patient samples.

The results revealed a significant and uniform reduction in PBL clock gene expression during the first week of ICU admission (FIG. 3). Bmall, the only gene not affected in endotoxin-challenge PBL, was also not reduced in PBL obtained from patients. Several genes, including Cryl, Per3, and Rora remained suppressed in the patients studied for at least an additional week during ICU admission (FIG. 3). The analysis indicates that the transient decline in circadian clock gene expression in PBL first noted during systemic inflammation induced by TLR4 activation (Haimovich et al. Crit Care Med 2010, 38:751-758) persists for an extended period in patients with injury induced systemic inflammation Circadian Clock Gene Expression in PBL:

The administration of a bolus dose of endotoxin to human subjects triggers well characterized acute phase inflammatory responses. All clinical symptoms were resolved in this model within 24 hours. This model was used to determine the effect of endotoxin relative to the expression of circadian clock genes in PBL.

Day Study:

Subjects were administered endotoxin (n=4) or saline (control; n=2) at 9AM (time 0). Blood samples were collected at the following times (in parenthesis are listed times relative to time 0), 9AM (-24 h), 2AM (-7 h), and 6AM (-3 h), on the day prior to the infusion, and 9AM (0 h), 12 PM (3 h), 3PM (6 h), 10PM (13 h), 2AM (17 h), and 9AM (24 h) post-infusion. Gene expression was analyzed by quantitative real-time (qRT) PCR. Unless otherwise indicated, data are expressed as mean fold change + SD relative to the infusion time, taken to be time 0.

It was found that the relative gene expression values determined at 9AM, 2AM or 6AM on the day prior to the infusion were close to, or above baseline for all genes. See FIG. 4A. As shown in the FIG., blood was collected at the indicated time points post-endotoxin (closed symbols) or saline (open symbols) infusion. Gene expression levels were analyzed by qRT- PCR and were expressed as fold change relative to the 9AM infusion time, taken to be time 0. The correlation between the time from infusion and the time-of-day is illustrated in the lower right hand corner. Data are expressed as mean fold change + SD.

It was found that the lowest relative gene expression level detected was that of Perl (0.7+0.29 at 2AM). Of the 10 genes examined, only Ror and Rev-erb showed significant time- dependent expression differences (P <0.01; one way ANOVA) with a peak at 6AM.

The infusion of endotoxin triggered a significant decline in Clock, Per3, Cryl, Cry2, Rora, CSNKle, and Rev-erb expression, reaching a nadir within 3-6 hours (FIG. 4A) (P < 0.0001 for all genes; one-way ANOVA). By 6 hours post-infusion, the expression values of the seven aforementioned genes had decreased by 80-90% relative to baseline. Perl and Per2 exhibited distinct expression patterns. Perl expression spiked within 3 hours and fell steeply thereafter, reaching a nadir at 13-17 hours post- infusion. Per2 exhibited a more modest endotoxin-induced response (P<0.001 one-way ANOVA). Bmall expression was not altered significantly post-endotoxin infusion (P>0.05, one-way ANOVA).

Next, the relative gene expression levels at 2AM (-7 h) pre-infusion to 2AM (17 h) post-infusion were compared. The differences between the gene expression levels pre- and post- infusion were statistically significant for all genes with the exception of Bmall. These findings indicate that the expression levels of nine out of the ten genes examined remained suppressed for at least 17 hours post-endotoxin challenge. Furthermore, the expression level of most genes remained at least two-fold lower than baseline levels at 24 hours post-challenge.

Plasma Cortisol and pro-inflammatory cytokines levels in endotoxin- and saline- challenged subjects were examined. As shown in FIGs. 4B-D, plasma Cortisol levels were monitored by direct radioimmunoassay and TNFa and IL-6 levels were determined by enzyme-linked immunoassay.

Blood plasma Cortisol levels in humans normally peak 3-4 hours after the end of the sleep/darkness period. Plasma concentrations of Cortisol, as well as the pro-inflammatory cytokines TNFa and IL-6, increase significantly in response to an endotoxin challenge. The anticipated increase in Cortisol concentrations was detected in all endotoxin subjects by 1.5 hours post-challenge. The Cortisol concentration peaked between 3-6 hours and returned to baseline levels at 24 hours (FIG. 4B). As previously reported in this model system, TNFa concentration peaked within 1-1.5 hours post- infusion and returned to baseline by 3 hours (FIG. 4C), whereas IL-6 concentration peaked within 2 hours and returned to baseline within 6 hours (FIG. 4D).

Night Study:

There is a possibility that the circadian/diurnal phase at the time of endotoxin challenge could influence the expression of clock genes in endotoxin-challenge PBL. Therefore, assays were carried out to examine volunteer subjects challenged with endotoxin (n=3) at 9PM.

Blood was collected at 9PM (-24), 2AM (-18 h), 6AM (-15 h), 10AM (-12 h), 2PM (-6 hr), and 6PM (-3h) on the day prior to the infusion, and at 9 PM (time 0), 10PM (1 h), 11PM (2 h), 12 Midnight (3 h), 1AM (4 h), 3AM (6 h), 6AM (9 h), 9AM (12 h), 2PM (18 h), 6PM (21 h), and 9PM (24 h) post-infusion. Consistent with the data presented in FIG, 5A for the day subjects, the relative gene expression levels of all ten genes were close to or above baseline during the 24 hours pre-infusion

As shown in FIG. 5, blood was collected at the indicated time points prior to endotoxin-infusion (open symbols) and post- infusion (closed symbols). Gene expression levels were analyzed by qRT-PCR and expressed as fold change relative to the 9PM infusion time, taken to be time 0. The correlation between the time from infusion and the time-of-day is illustrated in the lower right hand corner. It was found that several genes exhibited increased expression in the early hours of the day, but these differences were not statistically significant. Similarly, it was found that the expression of all ten genes remained above baseline levels post-saline infusion at 9PM (n=l) (data not shown). The infusion of endotoxin at 9PM triggered profound temporal changes in clock gene expression. The changes were remarkably similar to those seen in subjects challenged with endotoxin at 9AM. Clock, Per3, Cry 1 and Cry2, Rev-erb, Rora, and CSNKle expression decreased by approximately 80-90% in response to the endotoxin challenge (p<0.001) reaching their nadir between 4 and 9 hours post- infusion. As seen in the day subjects, the nadir in Perl expression was reached several hours after the nadir of most other genes. Per2 and Bmall were the least affected by endotoxin. The circulating Cortisol levels also increased significantly in endotoxin challenged subjects between 1.5- and 4-hours post-challenge, while the temporal changes in TNFa and IL-6 were indistinguishable from those observed in subjects challenged with endotoxin at 9AM. These observations establish that endotoxin suppresses the expression of circadian genes when administered at various times during the diurnal cycle.

Circadian Clock Gene Expression in PBL As Compared To Leukocyte Subpopulations:

Circadian clock gene expression was next compared among PBL, monocyte- and neutrophil-subpopulations (n=3 for each). Blood was obtained prior to endotoxin-infusion (time 0; 9AM), and 6- and 24-hours post-infusion.

As shown in FIG. 6, blood was collected at time 0, 6 and 24 hours post-endotoxin infusion. Total PBL, neutrophils and monocytes subpopulations were obtained. Gene expression levels were analyzed by qRT-PCR and expressed as fold change relative to the 9AM infusion time, taken to be time 0. The correlation between the time from infusion and the time-of-day is illustrated in the lower right hand corner. The data shown represent averages (n=3).

It was found that the expression patterns of Clock, Cry 1-2, Rora, Per 3, CSKNle, Reverb and Bmall noted in PBL were indistinguishable from those observed in neutrophils. In contrast, by 6 hours post-infusion the expression of Rora, Per3, and Rev-erb was suppressed in monocytes, while the expression of Clock, Cry-1, Cry-2, and CSNKle remained close to baseline levels. By 24 hours post- infusion, Clock, Cry-1, Cry-2, CSNKle, Per 3, Cryl and Cry2 gene expression levels were at least partially reduced in PBL, neutrophils, as well as monocytes. Perl expression increased in both neutrophils and monocytes by 6 hours of infusion. As observed in PBL, Perl and Bmall were the least responsive to endotoxin in neutrophils and monocytes.

These results indicate that the temporal changes in clock gene expression observed in PBL reflect changes that unfold in neutrophils, and to a lesser extent in monocytes. The results also indicate that the timing of the changes in clock gene expression triggered in response to endotoxin-infusion is leukocyte cell-type specific.

Endotoxin does not affect plasma melatonin's rhythms. The plasma melatonin levels in humans normally peak several hours before the end of the sleep/dark period. Analyses of plasma melatonin levels prior to infusion, and post-endotoxin or -saline infusion, revealed the anticipated increase in melatonin in the early hours of the day. See FIG. 7 A, where plasma melatonin concentrations were determined in blood samples obtained from eight control subjects at 10PM, 2AM, 6AM, 9AM and 10AM (open squares). Samples were also obtained at the indicated times of the day pre- and post-saline infusion ((n=l); open circles), and pre- and post-endotoxin infusion ((n=4); closed diamonds). Arrow indicates the 9 AM infusion time. As shown in FIG. 7B, samples were obtained at the indicated times of the day pre- and post-endotoxin infusion ((n=4); closed diamonds). Arrow indicates the 9PM infusion time. These observations indicate that endotoxin does not affect the apparent activity of the master clock during the acute phase of systemic inflammation.

Circadian clock genes exhibit highly related responses to endotoxin. To better define the relationship among circadian clock gene expression in endotoxin-challenged PBL, the gene expression data presented in FIGs. 4 and 6 were processed and clustered as described above. The results were shown in FIG. 8.

The gene expression data for each subject was cross-correlated across all studied genes within a subject. The cross-correlation data were normalized to one, where one represents a perfect correlation between gene responses. The correlation matrices for (A) endotoxin day subjects (n=4), (B) endotoxin night subjects (n=3), (C) endotoxin day and night subjects (n=7), or (D) placebo day and night subjects (n=3), were averaged and clustered into a tree form using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm with one minus the cross-correlation coefficient as a measure of similarity.

Shown in FIG. 8 are the combined day and night mean expression values of each gene post-endotoxin infusion. Where shown, bars represent standard error of the mean. The solid line that is featured in each panel of FIG. 8 was drawn based on the mean expression values of Clock, Cryl, Cry2, and Per3, which exhibited highly related responses to endotoxin in the day and night (FIG. 8) subjects. Co-clustering of the day and night expression data positioned CSKle and Rora in correlation with Clock, Per3, Cryl and Cry2 (FIG. 8 A). While the similarities in gene expression patterns seen in endotoxin- subjects were not observed in control subjects, clustering analyses of the combined day- and night-placebo data indicated correlations between Per2 and Bmall, and Cryl and Cry2. When the day and night endotoxin subjects data were analyzed as a group, the remarkably similar temporal decline in Clock, Cryl, Cry2, Per3, CSNKle, and Rora expression became apparent (FIG. 8).

The line featured in each of the panels in FIG. 5 represents the mean of Clock, Cryl, Cry2, and Per3 expression values. Perl, Per2, Rev-erb, and Bmall displayed distinct expression patterns as compared to the common response pattern of the remaining six genes. Perl expression spiked by 3 hours and declined thereafter reaching a nadir at 10-15 hours post- infusion. Per2 similarly reached its expression nadir 13-17 hours post-challenge. Reverb showed a slower and shallower decrease in expression, while the changes in Bmal expression were insignificant. The large number of clock genes exhibiting a similar endotoxin-induced response indicates that endotoxin is a potent entrainer of the circadian clock network in PBL.

The expression of many circadian clock genes is suppressed in PBL during the acute phase of systemic inflammation. The administration of a bolus-dose of endotoxin to human subjects triggers well-characterized acute inflammatory responses that are resolved within 24 hours. It was found that the expression of key genes implicated in the regulation of circadian clock function, including Clock, Per3, Cryl and Cry2, Rora, and Rev-erb, is decreased in PBL by 80-90% within 3-6 hours post-endotoxin infusion. The relationships among these genes, and/or their protein products, are characterized.

Clock and Bmall (Arntl; MOP3) regulate the transcription of Period (Per) and Cryptochrome (Cry) genes via E-box enhancer elements in their promoters. Per/Cry complexes relocate from the cytosol to the nucleus, where they function as Clock/Bmal repressors. Rev-erb and Ror are transcription factors that bind to the promoter region of Bmall to either suppress or enhance its expression, respectively. The expression of Rev-erb is in turn rhythmically regulated by the Clock/Bmal complex. The repressor Per and Cry proteins are phosphorylated by Casein kinase Ιε/δ (CSNKIe/5). The phosphorylated proteins are ubiquitinated and targeted for degradation by the proteosome. Once the Per and Cry proteins are degraded, the de-repressed Clock/Bmal may reinitiate their activity cycle. The precipitous declines in expression of multiple genes that act at various junctures in the circadian clock network indicate that clock activity in PBL is severely impaired during the acute phase of systemic inflammation.

Perl, Per2, Per3, and Bmall gene oscillations have been observed in human PBMCs. In one study, Perl and Per2 expression in PBMCs peaked in the early hours of the day, whereas Bmall peaked in the middle of the wake period. In another study, Per2 and BMAL1 cycled with a similar rhythm in PBMCs, while Rev-erb expression remained constant. Recently, the expression of ten circadian clock genes, including Per 1-3, Cryl and Cry2, Clock, and Bmall, was examined in PBMCs. Of the ten genes examined, only Per 1-3 showed rhythmic expression in most subjects, with no significant acrophase differences among the three genes. While prior studies aimed to uncover the rhythmicity of circadian clock genes in peripheral blood cells, the goal of this study was to determine the fate of clock gene expression in PBL during the acute phase of systemic inflammation. Hence, in contrast with the entrainment protocols used by others, the subjects descried in the examples described herein were not rigorously entrained by an extended sleep/wake schedule prior to or during the study phase. Despite the lack of sleep/wake entrainment, two sets of genes, Bmall and Per2 which according to one report cycle together in PBMSc, and Cryl and Cry2, exhibited correlated expression in PBL obtained from saline-infused subjects. Bmall and Per2 showed no correlated expression in subjects administered endotoxin. In contrast, Cryl and Cry 2, as well as Clock, Per3, CSNKle, and Rora exhibited correlated expression in PBL obtained from endotoxin subjects. Given the limited synchronization among clock genes and the significant inter- subjects variability previously noted in PBMCs obtained from sleep/wake entrained normal human subjects, the correlation observed among multiple circadian clock genes in PBL and neutrophils after endotoxin challenge indicate that endotoxin is a potent entrainer of the circadian clock network in PBL.

Melatonin, Cortisol, and Circadian Clock Gene Expression in PBLs during Acute Phase of Systemic Inflammation:

In humans, the activity of the central master clock is generally correlated with the secretion of melatonin, which is released in the early hours of the day. It was found that melatonin rhythms remained intact in endotoxin-challenged subjects, peaking in the early hours of the day. In contrast, as previously reported, endotoxin triggered a surge in plasma

Cortisol levels with a peak between 3-4 hours. Furthermore, while Cortisol levels peaked, many clock genes reached their expression nadir, such that the levels of Cortisol and clock gene expression became inversely related within three hours. These data indicate that centrally regulated plasma melatonin- and cortisol-rhythms, and PBL clock gene expression are independently regulated in response to endotoxin. These observations raise the possibility that the master clock and circadian clock gene expression in PBL become misaligned during the acute phase of systemic inflammation induced by endotoxin.

The results disclosed herein also indicate that endotoxin transiently suppresses the expression of clock genes in a peripheral tissue(s). Furthermore, the data described herein establish that in humans the significant perturbations in clock gene expression in PBL unfold while the rhythmicity of the master clock, determined based on plasma melatonin levels, appears to remain intact.

Pro-Inflammatory Cytokines and Clock Gene Expression in PBL:

Pro-inflammatory cytokines, which are released in the early stages of systemic inflammation, have been implicated in the regulation of circadian activity in mice. TNFoc- or IL-i -infusion suppressed the expression of several clock genes, including Per2 and Per3, in mice livers by binding to the E-box motives in their promoters. Clock and Bmall expression was not affected these cytokines. TNFoc levels surge in response to endotoxin, reaching an acrophase within 1.5-2 hours post-challenge. The expression of most clock genes examined in this study remained suppressed for up to seventeen hours.

Analyses of clock gene expression in human oral mucosa and skin revealed rhythmic expression of Perl, Cryl, and Bmall with acrophases in the early morning, late afternoon, and at night, respectively. No rhythmic expression of Clock was observed in these tissues. Boivin et al. (Blood 2003; 102: 4143-4145) were the first to examine circadian clock gene expression in human PBMCs. Analysis of PBL circumvents the need for cell-purification step(s), minimizes manipulation time, and hence is a practical approach for sample acquisition in the clinical setting. However, the use of a mixed cell population such as PBL introduces some level of uncertainty as the proportion of each immune cell type changes dynamically over-time post-endotoxin infusion with an early recruitment of neutrophils and a decrease in monocytes and lymphocytes counts. The blood cell type counts return to baseline within 12 hours post- treatment.

To address this potential limitation, clock gene expression was compared among PBL, monocytes, and neutrophils at select time points post-infusion in this study. The results revealed that the changes in clock gene expression observed in PBL were replicated in neutrophils. By 6 hours post-endotoxin infusion, several but not all clock genes, were also suppressed in monocytes. These findings establish that the changes in circadian clock gene expression observed in PBL are primarily reflective of changes occurring in neutrophils, and to a lesser extent in monocytes.

The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated herein in their entireties.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for determining whether a subject has, or is at risk of having, an inflammatory disorder, comprising

obtaining from the subject a sample; and

determining in the sample the expression levels of a plurality of genes, each gene being selected from (i) a first panel of up-regulated TLR4 and Injury Responsive (TIR) genes, (ii) a second panel of down-regulated TIR genes, or (iii) a third panel of core genes, whereby the subject is determined to have, or to be at risk of having, the disorder if:

(a) the expression level of each gene selected from the first panel is above a first predetermined reference value,

(b) the expression level of each gene selected from the second panel is below a second predetermined reference value, or

(c) the expression level of each gene selected from the third panel is below a third predetermined reference value.

2. The method of claim 1, wherein the inflammatory disorder is sepsis or a systemic inflammatory response syndrome.

3. The method of claim 1, wherein the sample contains leukocytes.

4. The method of claim 1, wherein the sample is a blood sample.

5. The method of claim 1, wherein the first, second, or third predetermined reference value is obtained from a control subject that does not have the disorder.

6. The method of claim 1, wherein the first panel of up-regulated TIR genes comprises those that are listed in Table 3 and whose expression levels in a healthy subject increase in response to an endotoxin.

7. The method of claim 1, wherein the second panel of down-regulated TIR genes comprises those that are listed in Table 3 and whose expression levels in a healthy subject decrease in response to an endotoxin.

8. The method of claim 1, wherein each gene is selected from the third panel of core genes.

9. The method of claim 8, wherein the third panel of core genes comprises the Cryl, Cry2, Per 3, Clock, Rora, Rev, CSNKle, and CDK4 genes.

10. The method of claim 9, wherein the plurality of genes are the Cryl, Cry2, Per3, Clock, Rora, Rev, CSNKle, and CDK4 genes.

11. A method for determining a prognosis of an inflammatory disorder in a subject that has received an injury, comprising

obtaining from the subject a sample; and

determining in the sample the magnitude of a change in the expression level of one or more genes, each gene being selected from (i) a first panel of up-regulated TLR4 and Injury Responsive (TIR) genes, (ii) a second panel of down-regulated TIR genes, (iii) a third panel of core genes, or (iv) a fourth panel of reversible responsive genes, whereby the magnitude is indicative of the prognosis of the subject.

12. The method of claim 11, further comprising comparing the magnitude to a predetermined reference value whereby the subject is determined to have a good prognosis if the magnitude is below the predetermined reference value.

13. The method of claim 12, wherein the predetermined reference value is obtained from a patient that has the disorder.

14. The method of claim 11, wherein the inflammatory disorder is sepsis or a systemic inflammatory response syndrome.

15. The method of claim 11, wherein the sample contains leukocytes.

16. The method of claim 11, wherein the sample is a blood sample.

17. The method claim 11, wherein the one or more genes are selected from the fourth panel.

18. The method of claim 17, wherein the sample is obtained from the subject within 12 days after receiving the injury or after the onset of the disorder.

19. The method of claim 18, wherein the sample is obtained from the subject within 9-12 days after receiving the injury or after the onset of the disorder.

20. The method claim 11 , wherein the one or more genes are selected from the first, second, or third panel.

21. The method of claim 20, wherein the sample is obtained from the subject 12 or more days after receiving the injury or after the onset of the disorder.

22. An array comprising a support having a plurality of unique locations, and any combination of:

(i) at least one nucleic acid having a sequence that is complementary to a gene selected from a first panel of up-regulated TLR4 and Injury Responsive (TIR) genes,

(ii) at least one nucleic acid having a sequence that is complementary to a gene selected from a second panel of down-regulated TIR genes,

(iii) at least one nucleic acids having a sequence that is complementary to a gene selected from a third panel of core genes, and

(iv) at least one nucleic acid having a sequence that is complementary to a gene selected from a fourth panel of reversible responsive genes,

wherein each nucleic acid is immobilized to a unique location of the support.

23. The array of claim 22, wherein the array comprises at least eight nucleic acids.

24. The array of claim 22, wherein the plurality of unique locations are selected from the group consisting of beads, spheres and optical fibers.

25. The array of claim 22, wherein the support comprises a material selected from the group consisting of: glass, coated glass, silicon, porous silicon, nylon, ceramic, and plastic.

26. A comprising

a probe having a nucleic acid sequence that is complementary to the sequence of a gene selected from (i) a first panel of up-regulated TLR4 and Injury Responsive (TIR) genes, (ii) a second panel of down-regulated TIR genes, or (iii) a third panel of core genes, or (iv) a fourth panel of reversible responsive genes, or

a pair of PCR primers for amplifying a mRNA of said gene.

27. The kit of claim 26, further comprising reagents for performing hybridization.

28. The kit of claim 26, further comprising reagents for performing PCR.

29. The kit of claim 26, wherein said gene is selected from the third panel of core genes.

30. The kit of claim 26, wherein the third panel of core genes comprises the Cryl, Cryl, Per 3, Clock, Rora, Rev, CSNKle, and CDK4 genes.

31. The kit of claim 26, wherein the kit comprises eight probes that are complementary to sequences of the Cryl, Cry2, Per3, Clock, Rora, Rev, CSNKle, and CDK4 genes, respectively.

32. The kit of claim 26, wherein the kit comprises eight pairs of primers for amplifying mRNAs of the Cryl, Cryl, Per3, Clock, Rora, Rev, CSNKle, and CDK4 genes, respectively.

33. The kit of claim 26, wherein the kit comprises the array of any of claims 22-25.