WO2012016030A2 - Détection d'une inflammation - Google Patents

Détection d'une inflammation Download PDF

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Publication number
WO2012016030A2
WO2012016030A2 PCT/US2011/045705 US2011045705W WO2012016030A2 WO 2012016030 A2 WO2012016030 A2 WO 2012016030A2 US 2011045705 W US2011045705 W US 2011045705W WO 2012016030 A2 WO2012016030 A2 WO 2012016030A2
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genes
panel
gene
subject
endotoxin
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PCT/US2011/045705
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WO2012016030A3 (fr
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Stephen F. Lowry
Beatrice Haimovich
Michael Reddell
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University Of Medicine And Dentistry Of New Jersey
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • This invention relates to reagents and methods for detecting inflammation in a subject.
  • 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.
  • PAMPs pathogen-associated molecular pattern(s)
  • 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.
  • changes in cytokines are significantly less robust, and hence difficult to establish, during conditions of low-grade inflammation.
  • 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.
  • TLR4 Toll-like receptor4 pathway
  • 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.
  • 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.
  • 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.
  • this panel consists of the Cryl, Cryl, Per 3, Clock, Rora, Rev, CSNKle, and CDK4 genes.
  • 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.
  • an inflammatory disorder e.g., sepsis or a systemic inflammatory response syndrome
  • 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.
  • the one or more genes for practicing the prognosis method are selected from the fourth panel.
  • the sample can be obtained from the subject within 12 days after receiving the injury or after the onset of the disorder.
  • the sample is obtained from the subject within, e.g., 9-12 days after receiving the injury or after the onset of the disorder.
  • the one or more genes are selected from the first, second, or third panel.
  • 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.
  • 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.
  • 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.
  • the array has at least eight (e.g., 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,
  • the support can contain a material selected from the group consisting of glass, coated glass, silicon, porous silicon, nylon, ceramic, and plastic.
  • 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.
  • 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.
  • 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.
  • the kit contains one or more of the above- mentioned arrays.
  • 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.
  • 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,
  • FIG. 3A shows the mean fold change in gene expression observed in the PBLs, where error bars are SEM.
  • 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.
  • 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.
  • 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.
  • 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 identified are involved in TLR4 signaling. Based on their expression patterns, these genes can be divided into two groups.
  • 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.
  • 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.
  • PAMPs pathogen-associated molecular patterns
  • DAMPs damage-associated molecular patterns
  • HMGBl High-mobility group box 1
  • HSP heat shock proteins
  • ROS reactive oxygen species
  • 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.
  • rhythm genes that are suppressed in PBL from both endotoxin challenged subjects and ICU patients are circadian clock genes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • test sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • 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.
  • RNA samples can be isolated from biological samples using any of a number of well-known procedures.
  • biological samples can be lysed in a guanidinium-based lysis buffer, optionally containing additional components to stabilize the RNA.
  • 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.
  • 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).
  • 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.
  • RNAse inhibitors are optionally added to the crude samples.
  • genomic DNA can contribute one or more copies of a target sequence, e.g., a gene, depending on the sample.
  • 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.
  • one of the two target- specific primers could be designed to span a splice junction, thus excluding DNA as a template.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibody bears a detectable label.
  • label refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, 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.
  • 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
  • detectable substance or labels include radio isotopes (e.g., I,
  • I, S, H, or P enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or ⁇ -glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., QdotTM nanoparticles by the Quantum Dot Corporation, Palo Alto, CA).
  • enzymes e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or ⁇ -glactosidase
  • fluorescent moieties or proteins e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP
  • luminescent moieties e.g., QdotTM 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.
  • 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.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • an immune-suppressant e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • 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.
  • 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.
  • organ or tissue transplantation is often accompanied by nonspecific immune suppression therapy to prevent T cell-mediated rejection.
  • these immunosuppressants can cause infection, hypertension, cancer, and other undesirable side effects. Therefore, there is a need for monitoring the suppression.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • 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.
  • the biochip may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol 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.
  • biotinylated oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • probes may be synthesized on the surface using techniques such as photopolymerization and photolithography.
  • 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.
  • an expressed transcript (e.g., a transcript of a gene described herein) is represented in the nucleic acid arrays.
  • 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.
  • the nucleic acid arrays of the invention can have one probe specific to each target gene or exon.
  • 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).
  • the array may contain probes tiled across the sequence of the longest mRNA isoform of a gene.
  • 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.
  • kit may include instructional materials containing directions (e.g., protocols) for the practice of the methods described herein.
  • the kit may be a kit for the amplification, detection, identification or quantification of a target mRNA sequence.
  • the kit may contain a poly(T) primer, a forward primer, a reverse primer, and a probe.
  • 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.
  • 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.
  • at least one included polynucleotide sequence corresponds to a control sequence, e.g., a "housekeeping" gene, ⁇ -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.
  • kits that are suitable for amplifying nucleic acid corresponding to the expressed RNA samples are provided.
  • a kit includes reagents and primers suitable for use in any of the amplification methods described above.
  • 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).
  • kits one or more materials and/or reagents required for preparing a biological sample for gene expression analysis are optionally included in the kit.
  • 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.
  • 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.
  • the methods and kits described in the present invention allow quantitation of other products of gene expression, including tRNA, rRNA, or other transcription products.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject is a human.
  • the subject is an experimental, non-human animal or animal suitable as a disease model.
  • 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.
  • 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.
  • 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.
  • inflammatory dermatoses e.g., dermatitis, eczema, atopic derma
  • 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.
  • ITP thrombocytopenia
  • AHA autoimmune hemolytic anemia
  • SLE systemic lupus erythematosus
  • gene 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • probe 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 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.
  • 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.
  • temperature such as salt concentration
  • the term "reference value” refers to a value that statistically correlates to a particular outcome when compared to an assay result.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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 TRIzolTM solution (SIGMA, St. Louis MO), sheared 10 times with a 18 gauge needle, and frozen at -70°C.
  • TRIzolTM solution SIGMA, St. Louis MO
  • First strand cDNA synthesis was performed using reverse transcription (SUPERSCRIPTII, INVITROGEN, Carlsbad, CA) in a reaction containing 5 ⁇ g 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).
  • 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).
  • Steps outlined in this section were performed by the microarray core facility at UMDNJ. Following fragmentation of the biotinylated cRNA, 15 ⁇ g was placed in hybridization cocktail, heated to 95° C, centrifuged and then hybridized to the FocusTM GeneChip microarray (AFFYMETRIX) for 16 hours at 45 °C. Chips were then washed, stained with streptavidin phycorerythrin and scanned on the Agilent Gene Array ScannerTM (AGILENT TECHNOLOGIES, Santa Clara, CA).
  • AFFYMETRIX FocusTM GeneChip microarray
  • GeneSpringTM 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 AnalysisTM (INGENUITY, Palo Alto, CA) as previously described. The microarray data have been submitted to Gene Expression Omnibus (accession number pending).
  • qPCR quantitative real-time polymerase chain reaction
  • B2M beta-2 -micro globulin
  • 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% NH 4 C1, 0.1% KHC0 3 , 0.0037% Na 4 EDTA in H 2 0) 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.
  • TRIZOL solution INVITROGEN, Carlsbad, California
  • RosetteSepTM 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.
  • 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).
  • FITC CD66b-fluoroscein isothiocynate
  • PE CD2-phycoerythrin
  • BD BIOSCIENCES CD33-peridinin-chlorophyll-protein complex
  • cyanin dye 5.5 PerCP-Cy5.5 cyanin dye 5.5 PerCP-
  • 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.
  • 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.
  • UPMA Unweighted Pair Group Method with Arithmetic Mean algorithm
  • 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.
  • Cytokine levels were determined by sandwich ELISA using monoclonal/polyclonal antibody pairs (R & D SYSTEMS, Minneapolis, MN) (additional information) and the vendor's protocol.
  • 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.
  • 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.
  • 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.
  • TIR TLR4 and injury responsive genes
  • KIR2D domains short cytoplasmic tail, 1 /tail, 2 / tail, 4
  • TBP binding protein
  • 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.
  • 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.
  • the TIR genes group includes 273 down-regulated and 176 up-regulated genes (Table
  • 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.
  • EIF/EEF genes EIF3E, EIF4A2, and EEF1A1
  • HNRNP genes HNRNPA1, HNRNPA1///LOC7, HNRNPH1, HNRNPH2///RPL3, HNRNPR, and HNRPDL
  • HNRNPA1, HNRNPA1///LOC7, HNRNPH1, HNRNPH2///RPL3, HNRNPR, and HNRPDL HNRNPA1, HNRNPA1///LOC7, HNRNPH1, HNRNPH2///RPL3, HNRNPR, and HNRPDL
  • 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.
  • TIDBase a web-based public resource supported by the type 1 diabetes (TID) research community (www.tldbase.org), identified more than 1400 Myorelated interactions.
  • TIDBase a web-based public resource supported by the type 1 diabetes (TID) research community (www.tldbase.org), identified more than 1400 Myorelated interactions.
  • TIDBase a web-based public resource supported by the type 1 diabetes (TID) research community (www.tldbase.org), identified more than 1400 Myorelated interactions.
  • TIDBase a web-based public
  • PFK-2 6-phosphofructo-2-kinase
  • 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.
  • PDK pyruvate dehydrogenase kinase
  • 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.
  • Rora one of the key regulators of the circadian clock.
  • 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.
  • qRT quantitative real-time
  • 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.
  • TNFa concentration peaked within 1-1.5 hours post- infusion and returned to baseline by 3 hours (FIG. 4C)
  • IL-6 concentration peaked within 2 hours and returned to baseline within 6 hours (FIG. 4D).
  • 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.
  • Circadian clock genes exhibit highly related responses to endotoxin.
  • 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.
  • UPMA Unweighted Pair Group Method with Arithmetic Mean
  • FIG. 8 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).
  • 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.
  • 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.
  • circadian clock genes 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.
  • 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 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 ⁇ / ⁇ (CSNKIe/5). The phosphorylated proteins are ubiquitinated and targeted for degradation by the proteosome.
  • 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.
  • Perl and Per2 expression in PBMCs peaked in the early hours of the day, whereas Bmall peaked in the middle of the wake period.
  • Per2 and BMAL1 cycled with a similar rhythm in PBMCs, while Rev-erb expression remained constant.
  • 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.
  • Cryl and Cry 2 as well as Clock, Per3, CSNKle, and Rora exhibited correlated expression in PBL obtained from endotoxin subjects.
  • Clock Per3, CSNKle, and Rora exhibited correlated expression in PBL obtained from endotoxin 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.
  • 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.
  • 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.
  • 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.
  • clock gene expression was compared among PBL, monocytes, and neutrophils at select time points post-infusion in this study.

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Abstract

La présente invention concerne des procédés et des réactifs de détection et de surveillance d'une inflammation et d'une lésion chez un sujet par détermination des niveaux d'expression de groupes de gènes.
PCT/US2011/045705 2010-07-28 2011-07-28 Détection d'une inflammation WO2012016030A2 (fr)

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DE102014112923A1 (de) 2014-09-09 2016-03-10 Friedrich-Alexander-Universität Erlangen-Nürnberg Verfahren und System zur Detektion von Krankheitsbildern
DE102014112924A1 (de) 2014-09-09 2016-03-10 Friedrich-Alexander-Universität Erlangen-Nürnberg Verfahren und System zur Detektion von Krankheitsbildern
EP3519594B1 (fr) * 2016-09-29 2022-09-07 Secretary of State for Health and Social Care Dosage pour distinguer un sepsis d'un syndrome de réponse inflammatoire systémique

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102014112923A1 (de) 2014-09-09 2016-03-10 Friedrich-Alexander-Universität Erlangen-Nürnberg Verfahren und System zur Detektion von Krankheitsbildern
DE102014112924A1 (de) 2014-09-09 2016-03-10 Friedrich-Alexander-Universität Erlangen-Nürnberg Verfahren und System zur Detektion von Krankheitsbildern
EP3519594B1 (fr) * 2016-09-29 2022-09-07 Secretary of State for Health and Social Care Dosage pour distinguer un sepsis d'un syndrome de réponse inflammatoire systémique

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