JP2007522800A - Evaluation method of tissue inflammatory response using expression profile of endothelial cells - Google Patents

Abstract

  The present invention is a method for assessing a tissue inflammatory response, comprising the step of quantitatively measuring the level of at least five transcripts shown in Table 1 or the protein encoded thereby in a sample; and Comparing the amount of the transcript or protein thus measured to the level of the transcript obtained from a control sample. Also provided are methods for the diagnosis of symptoms associated with tissue inflammatory responses, as well as gene chip arrays and protein-based assays suitable for use in these methods. Assay methods for determining modulators of tissue inflammatory responses or symptoms associated therewith also form part of the present invention.

Description

  The present invention relates to gene expression profiles of endothelial cells and the use of these profiles in the evaluation and treatment of disease symptoms.

  Leukocyte migration into tissues is an important part of the normal function of the immune system, mucosal surface, endometrium, mammary gland and ovary. On the other hand, migration of leukocytes from the vascular lumen into the tissue can also be stimulated by an inflammatory response. Inflammation is the body's response to injury or infection and typically involves the movement of leukocytes and proteins that pass through the endothelial wall and enter the tissue.

  Many signaling molecules are involved in the inflammatory response. Some of these act on the endothelium so that the endothelial cells do not adhere too tightly to each other and / or make their surfaces adhere to the passing white blood cells. Other signals act as attractants for leukocytes or promote the expression of adhesion molecules on leukocytes. Yet other signals regulate leukocyte activation and survival.

  Signaling molecules include “quick release” mediators that are involved in increasing vascular permeability, such as histamine. They act synergistically with later stage “process induced” mediators. These later-stage mediators include chemokines that attract leukocytes into the inflamed area, and cytokines that are later released from inflammatory and activated endothelial cells.

  TNF-α, IL-1β and IL-8 are inflammatory mediators found at high concentrations at sites of inflammation, and endothelial cells have receptors for these mediators (1-3).

  TNF-α is a primitive cytokine initially isolated from macrophages, but is now by various cells including lymphocytes, monocytes, neutrophils, T cells, myocytes and smooth muscle cells. It is known to be generated. In a wide variety of cell types, TNF-α encounters TNF receptor 1 to initiate apoptosis. Receptor binding also causes sustained NF-κB activation, which is responsible for the expression of a range of genes involved not only in the inflammatory response but also in enhanced cell survival and enhanced resistance to TNF-α-induced apoptosis. is necessary. Signaling pathways that are also activated in endothelial cells by TNF-α include phosphatidylinositol (PI) kinase-dependent kinases, stress-activated protein kinases (SAPK), and mitogen-activated protein kinases (MAPK) (3, 4) .

  IL-1β is another primitive cytokine. The primary source of secreted IL-1β is monocytes, which are mainly activated macrophages, neutrophils, platelets, T cells and B cells, natural killer (NK) cells, endothelial cells, SMCs (Smooth muscle cells) as well as a wide variety of other cell types including fibroblasts. IL-1β is synthesized as a precursor and, when cleaved by proteolysis, can activate a number of cell types that have a role in immunity and inflammation. As with TNF-α, IL-1β activates the NF-κB (nuclear factor κB) survival pathway and the MAPK cascade p42 / p44, p38 and Jnk (5).

  IL-8 is a chemokine that is closely associated with angiogenesis. The main sources of IL-8 are monocytes, neutrophils, T cells, NK cells, endothelial cells, fibroblasts and epithelial cells. IL-8 is involved in the process of leukocyte migration into tissues and other aspects of the inflammatory response. Its production is not constitutive and is induced by proinflammatory cytokines. In vitro, IL-8 binds to heparin and induces endothelial cell migration, proliferation and survival (6). IL-8 and its receptor appear to be critically important for angiogenesis and tumor progression (7, 8).

  Inflammatory molecular mediators such as TNF-α, IL-1 and IL-8 act on target cells in inflamed tissues such as endothelial cells, fibroblasts and leukocytes. For example, the effects of a combination of inflammatory mediators such as these three are synergistic. That is, the “cooperative” action of these factors is often different from the sum of their individual effects.

As typically seen in inflammatory diseases, the inflammatory response of a tissue is typically accompanied by abnormal or excessive migration of leukocytes and proteins into the tissue. This can cause a number of acute or chronic inflammatory disorders. Leukocyte and protein migration may play some role even in situations where tissue is not an inflammatory disease but the tissue shows an inflammatory response. For example, atherosclerosis and endometriosis can be caused by migration of leukocytes to the lesion. Repair of tissue necrosis that occurs in a stroke or heart attack also requires an inflammatory response. Tumor growth may be regulated by leukocytes that migrate from blood vessels into the tumor. These leukocytes release factors that directly promote tumor cell growth and tumor angiogenesis. In addition, leukocytes that migrate into the tumor mediate an immune response to the tumor cell-specific antigen and subsequent anti-tumor effects. Thus, the role of inflammation in tumor growth is complex and involves an equilibrium between the promoting and inhibitory pathways.
Schraufstatter, IU, Chung, J., and Burger, M. (2001) IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways. American Journal of Physiology 280, L1094-L1103 Dinarello, CA (1998) Interlukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Intern Rev Immunol 16, 457-499 Aggarwal, BB (2000) Tumour necrosis factor receptors associated signaling molecules and their role in activation of apoptosis, JNK and NF-kB. Annals of Rheumatic Disease 59 (suppl), i6-i16 Madge, LA, and Pober, JS (2001) TNF Signaling in Vascular Endothelial Cells.Experiment and Molecular Pathology 70, 317-325 O'Neill, LAJ, and Dinarello, CA (2000) The IL-1 receptor / toll-like receptor superfamily: crucial receptors for inflammation and host defense.Immunology Today 21, 206-209 Li, A., Dubey, S., Varney, ML, and Singh, RK (2002) Interleukin-8-induced proliferation, survival, and MMP production in CXCR1 AND CXCR2 expressing human umbilical vein endothelial cells.Microvascular Research 64, 476- 481 Strieter, RM, Polverini, PJ, Arenberg, DA, Walz, A., Opdendakker, G., Van Damme, J., and Kunkel, SL (1995) Role of CXC chemokines as regulators of angiogenesis in lung cancer.Journal of Leukocyte Biology 57, 752-762 Kitadai, Y., Haruma, K., Sumii, K., Yamamoto, S., Ue, T., Yokozaxi, H., Yasui, W., Ohmoto, Y., Kajiyama, G., Fidler, IJ, and Tahara, E. (1998) Expression of interleukin-8 correlates with vascularity in human gastric carcinomas.American Journal of Pathology 152, 93-100

  Given the role of inflammatory responses in a wide variety of pathologies, there remains a need to develop methods for assessing or treating human pathologies associated with such responses. Inflammatory responses are also an essential feature of physiological processes such as female fertility and wound healing. In order to modulate these processes, a method for modulating the inflammatory response is needed.

  The present inventors analyzed changes in the amount of transcripts produced when human umbilical vein endothelial cells (HUVEC) were contacted with a mixture of TNF-α, interleukin-1β and interleukin-8. These factors are typically present at elevated concentrations in tissues during the inflammatory response and are secreted by a wide variety of cell types over time.

  To obtain a consensus set of transcripts that are regulated in endothelial cells of multiple individuals during an inflammatory response, primary cell cultures of endothelial cells from a number of different individuals were tested. Data were analyzed using a novel combination of bioinformatics algorithms including a modified Loess standardization and CyberT algorithm.

  We also analyzed expression in various endothelial cell types obtained from various parts of the body, namely HUVEC, human coronary artery endothelial cells (HCAEC) and human uterine microvascular endothelial cells (UtMVEC). A number of transcripts have been found to be consistently regulated by inflammatory signals in all three cell types, including the most strongly regulated transcripts in HUVEC.

  Thus, in the present invention, we use a novel methodology to elucidate for the first time a pattern of changes in the regulation of the expression of certain genes in endothelial cells in response to inflammatory signals, For the first time revealed a consensus set of genes whose transcripts are affected in response to inflammatory signals in endothelial cells derived from and thus have broad applicability to the patient population.

  Thus, the present invention provides a means for evaluation (eg, diagnosis, prognosis or monitoring) of tissue inflammatory responses or symptoms associated therewith. Such a response is typically associated with one of a number of symptoms, which may include leukocyte migration across the endothelium. Thus, by measuring a tissue inflammatory response, it is also possible to measure symptoms that the response may be associated with. Also, the finding that multiple transcripts of both genes known per se and genes known from ESTs are regulated in these responses and thus in the symptom is the provision of new assay targets for such symptom and new Enables the development of treatments.

Accordingly, in a first aspect, the present invention is a method for assessing a tissue inflammatory response comprising:
Making a quantitative measurement of the level of at least five transcripts shown in Table 1, or the protein encoded thereby, in a sample;
Comparing the amount of the transcript or protein thus measured to the level of the transcript or protein obtained from a control sample of cells.

  Preferably, the sample comprises cells taken from a site suspected of being affected by the patient's tissue inflammatory response, preferably endothelial cells, or patient blood, serum or urine.

  In another embodiment, the present invention is a gene chip array suitable for use in the above method of the present invention, comprising at least 5 nucleic acids suitable for detection of at least 5 transcripts shown in Table 1. Providing an array comprising optionally a control specific for the transcript; and optionally at least one control for the gene chip.

  In yet another aspect, the invention is a protein-based assay suitable for use in the method of the invention, comprising at least 5 proteins encoded by transcripts comprising those shown in Table 1; Optionally provides a control specific for the protein; and optionally an assay for evaluation of at least one control for the assay.

In yet another aspect, the present invention provides an assay method for determining a modulator of a tissue inflammatory response or associated condition comprising:
a) providing a protein encoded by the transcript from Table 1;
b) contacting the protein with a candidate modulator of its activity;
c) determining whether the candidate modulator is capable of modulating the activity of the protein, or a) providing cultured endothelial cells;
b) contacting the cell with a candidate modulator of the tissue inflammatory response;
c) determining whether the candidate modulator is capable of modulating the level of at least one transcript selected from the transcripts of Table 1.

  The modulator obtained by such a method can be used in a method of modulating a tissue inflammatory response in a human or animal subject, eg, to treat the symptoms described below.

  In another embodiment, the identification of genes of unknown function and genes of ESTs allows the development of new potential targets for therapeutic intervention. Accordingly, the present invention provides a vector comprising a sequence encoding a transcript from Table 1b operably linked to a promoter for transcription of the sequence. Such vectors are useful in the expression of proteins that can be therapeutic agents or therapeutic targets. The vector itself may also have direct therapeutic uses, for example in gene therapy applications.

Table Description Table 1a lists transcripts of genes with previously recognized functions whose levels are regulated in endothelial cells by the inflammatory mediators TNFα, IL-1β and IL-8.

  Table 1b lists transcripts of genes or ESTs that do not have an already recognized function whose levels are regulated in endothelial cells by the inflammatory mediators TNFα, IL-1β and IL-8.

  Table 2a provides the nucleic acid sequence for each of the probes shown in Table 1a.

  Table 2b provides the nucleic acid sequence for each of the probes shown in Table 1b.

Detailed Description of the Invention References to Tables 1 and 2, respectively, refer to both Table 1a or 1b, respectively, unless it is clear from the context to refer to only one of the components a or b of Table 1 or Table 2, respectively. Or it should be construed to refer to both Tables 2a and 2b.

Symptoms Related to Tissue Inflammatory Response The present invention, in its first aspect, relates to a method for evaluation of a tissue inflammatory response. As noted above, it is generally recognized that leukocytes are involved in the inflammatory response of tissues. The inflammatory response may involve the migration of white blood cells through the endothelium. On the other hand, proteins involved in such inflammatory responses may cause migration of leukocytes to inflamed tissues, may be involved in or cause leukocyte activation, endothelial cell activation and angiogenesis, or leukocytes or Inhibiting endothelial cell apoptosis may affect the inflammatory response.

  The tissue inflammatory response is typically associated with the patient's condition or condition. As used herein, “symptom” means any pathologic or bodily response that is involved in, causes or is associated with a tissue inflammatory response.

  The tissue inflammatory response can be excessive or undesirable, eg, as in the case of inflammatory diseases as described below, or for example leukocyte adhesion deficiency types 1 and 2 (LAD-1 and LAD-2) As in the case of symptoms associated with abnormal adhesion proteins such as may be insufficient.

  Tissue inflammatory response and transendothelial leukocyte migration alter changes in endothelial cells (eg changes in adhesion molecule expression) that alter the ability of leukocytes to adhere to endothelial cells, or change chemokine-mediated recruitment of leukocytes to inflamed tissues Can be affected by. In the present invention, it is preferred that the changes in the endothelial cells are responsive to inflammatory signals.

  Symptoms assessed by the methods of the present invention are such that endothelial cells at the site of or affected by the inflammatory response promote the inflammatory process and allow migration of leukocytes across the endothelium. It can be changed.

  Typically, leukocyte migration across the endothelium occurs as a response to inflammatory signals. Thus, the tissue inflammatory response evaluated by the method of the present invention is one that produces an inflammatory signal. Typical examples of such symptoms include inflammatory diseases, vasculitis syndrome, atherosclerosis and related diseases, symptoms associated with tumor growth, and chronic transplant rejection.

  Inflammatory diseases include inflammatory bowel disorder, psoriasis, ischemia reperfusion, adult respiratory distress syndrome, asthma, allergic rhinitis, dermatitis, meningitis, encephalitis, uveitis, leukocyte emigration, central Nervous system inflammatory disorder, Alzheimer's disease, endometriosis, multiple sclerosis, multiple organ injury syndrome, alcoholic hepatitis, bacterial pneumonia, antigen-antibody complex mediated disease, lung inflammation (pleurisy, alveolitis, Including pneumonia, chronic bronchitis, bronchiectasis, cystic fibrosis and COPD), vasculitis, nodular polyarteritis, giant cell arteritis, microscopic polyarteritis, preeclampsia and autoimmunity Diseases (eg, rheumatoid arthritis, Sjogren's syndrome, and multiple forms of renal failure due to autoimmune attack primarily on endothelial cells in the glomeruli).

  In atherosclerosis and related diseases, such as coronary heart disease, peripheral vascular disease and cerebrovascular stroke, leukocyte migration through the endothelial layer and into the vascular wall of the atherosclerotic plaque site is associated with leukocytes and substrata associated with the plaque. Driven by inflammatory signals from endothelial cells. Plaque instability and concomitant rupture leading to thrombosis and vascular stenosis or occlusion may be associated with inflammatory effects on endothelial cells within the plaque area.

  Tumor growth can be regulated by leukocytes that migrate from blood vessels into the tumor and release factors that directly promote tumor cell proliferation and tumor angiogenesis. Thus, cancer and especially cancer with solid tumors may also be suitable therapeutic indications.

  Chronic transplant rejection, as important, includes immune cell migration through blood vessels and immune attack on endothelial cells and is therefore suitable for evaluation by the methods of the present invention.

  These and other symptoms associated with the release of inflammatory signals can be the subject of the methods of the present invention.

Evaluation of Tissue Inflammatory Response In the present invention, the measurement of “collected from a site” that is considered to be affected by the tissue inflammatory response is based on an in vitro method performed on a sample taken from the body. It will be understood that The step of taking the sample (eg, by biopsy) is not part of the invention itself.

  In addition to or instead of techniques such as tissue biopsy, body fluids such as blood, serum and urine can be collected to reveal inflammation-induced changes in these body fluids. Such a change can be indicated, for example, by a change in the amount of endothelial cell-derived protein encoded by the sequence of Table 1 in the sample.

  Accordingly, the present invention is a method for diagnosing symptoms associated with a tissue inflammatory response, wherein at least one of the transcripts of Table 1 in a sample from a patient suspected of being affected by such symptoms. A method comprising measuring the amount of endothelial cell-derived protein encoded by the five species is provided.

  As described above, the data obtained in the present invention is based on the fact that contact between endothelial cells and TNFα, IL-1β, and IL-8 is partly transcribed in consideration of variation between individuals and between different endothelial tissues. It shows that it causes a characteristic change to the product. These changes can take the form of patterns of response or can affect individual transcripts. Therefore, it is possible to use these characteristic changes as a standard for diagnosing the symptoms as described above.

  In addition to or instead of diagnosis, symptom assessment can be a determination of the severity of symptoms or the exact clinical subtype. It can also be a method of prognosing the future course of symptoms. Furthermore, the evaluation can be performed over a period of time, for example in the case of monitoring treatment of symptoms.

  The invention, in its first aspect, assesses tissue inflammatory response by comparing transcript levels in a sample taken from a patient exhibiting an inflammatory response to transcript levels of the same gene in a control sample. Provide a method.

  As mentioned above, the sample can be a sample of cells (eg, endothelial cells) from a particular tissue exhibiting an inflammatory response, or it can be a protein-containing body fluid, such as blood, serum or urine.

  A control sample for the method is typically a sample of unaffected cells, i.e. cells derived from tissue that does not exhibit a tissue inflammatory response, or cells that are not believed to be affected by the condition. is there. Both the test sample and the control sample are preferably of the same cell type, more preferably the test sample and the control sample are of endothelial cells. Most preferably, the test sample and the control sample are of the same endothelial cell type, i.e. from the same tissue location.

  If necessary, control samples can be taken from the subject population. When using a population of subjects, it is possible to make a comparison with the average (eg, mean or median) transcript level in a sample of cells from the population. Affected tissue can also be used as a control. Typically, the affected tissue is from a patient other than the patient from whom the test sample was taken. When diseased tissue is used as a control, it is generally preferred that the patient from whom the sample is taken has or is thought to have the same symptoms as the patient from whom the test sample was taken.

  In some embodiments, the methods of the invention can include the use of more than one control. For example, the sample to be tested can be compared to the normal or unaffected sample from the same patient or to the transcript level of a diseased sample from another patient.

  As mentioned above, the method of the present invention allows monitoring of symptoms either before, during or after treatment. If the test sample is from a patient with or suspected of having tissue inflammation, it is generally preferred to collect the sample at an earlier time point to obtain a medical history.

  In another embodiment, the method allows an assessment of the effectiveness of a particular treatment. By comparing the severity of tissue inflammation at two or more time points in the same patient, it is possible to determine whether a particular treatment regimen has a positive effect. The effectiveness of one prescription may vary from patient to patient or during the course of the disease.

  In a preferred embodiment, the present invention is practiced by examining the levels of multiple gene transcripts or multiple proteins derived from these transcripts. This is because the present inventors have found that transcript levels of individual genes can vary between individual patients. It is therefore desirable to assess transcript levels for several genes. For example, it is possible to assess at least 5, preferably at least 10, more preferably at least 20 transcripts of Table 1 or proteins derived from the transcripts of Table 1.

  The level of expression can be measured individually or as a specific combination for a gene or protein, or the pattern of expression of multiple genes can be measured.

  In the first aspect of the invention, the level of transcript evaluated is compared in a test sample and a control sample. A response to an inflammatory signal can increase or decrease transcript levels. Thus, there can be up-regulation or down-regulation of transcripts in response to inflammatory signals. Whether there is either up-regulation or down-regulation is not critical to the present invention, but rather it is important that a change from the control occurs.

  As an aid to measuring differences in transcript or protein regulation between test and control samples, it is possible to measure the amount of up-regulation or down-regulation. This can be expressed as an “magnification” of increase or decrease. It is not important to the present invention how much the increase or decrease in transcript level is, but rather it is important that a significant change from the control occurs. In the present invention, it is generally preferred that at least a 2-fold change (ie, increase or decrease) in transcript level occurs. Although there is no upper limit to the amount of increase or decrease in transcript or protein levels, we have observed a 50-fold change in the level of some transcripts and an apparent de novo upregulation for some proteins. Found that can be shown.

  Furthermore, in evaluating transcript or protein level up-regulation or down-regulation, it is not necessary for all transcripts or proteins to be assessed to be regulated in the same amount. More important in this regard is the establishment of a pattern of regulation.

  In one embodiment of the invention, the transcript or protein to be evaluated comprises one or more transcripts of one or more protein families or groups. Preferably, the method comprises at least one transcript or protein in each of such protein families or groups, preferably in more than one, preferably 3, 4, 5 or more, more preferably in each of such protein families or groups. Including measuring the level.

  Typical such groups or families include proteases, bone morphogenetic proteins, cell signaling proteins, proteins involved in transcriptional regulation, enzymes, cell cycle regulatory proteins, intercellular communication proteins, nuclear proteins, complement proteins, extracellular matrix proteins , Proteosome, heavy metal binding protein, TNF receptor superfamily protein, HLA protein, cell adhesion protein, cytokine / chemokine, cytokine / chemokine receptor, cytoskeletal protein, apoptosis regulator, growth factor and growth factor receptor.

  The transcript or protein to be evaluated includes one or more selected from cytokine / chemokine, cytokine / chemokine receptor, cytoskeletal protein, extracellular matrix protein, apoptosis regulator, growth factor and growth factor receptor, Generally preferred.

  Of particular interest in the methods of the present invention is the regulation of specific transcripts or proteins. These can be evaluated as such or as part of a pattern of regulation. Therefore, in the method of the present invention, the transcript or protein to be evaluated is colony stimulating factor 3 (granulocyte), colony stimulating factor 2 (granulocyte-macrophage), granulocyte chemotactic protein 2, diubiquitin, ELAM. -1, TNF inducible protein 6, Exodos1, IL-1β, VCAM-1, ICAM-1, IAP1, TNF inducible A20, RIPK2, MMP 10, TRAF1, JAK binding protein, bispecific phosphatase 4, IL- 6, IL-8, Gro-γ, MCP-1, Gro-β, Gro-α, ENA-78, fractalkine, low molecular weight inducible cytokine subfamily A14, low molecular weight inducible cytokine A5, LI7 receptor, toll / Interleukin 1 receptor-like 4, CCAAT, MAD-3, CO -2, Mn SOD, NO synthase, L-kynurenine hydrolase, tissue factor pathway inhibitor 2, laminin γ2, Toll-like receptor 2, natural killer cell transcript 4, TNFα-inducible protein 2, cationic amino acid transporter 2A, fatty acid Preferably, it comprises one or more of binding protein 4, TNF receptor superfamily member 11b and TNF superfamily member 9.

  In the method of the present invention, the transcript or protein to be evaluated is colony stimulating factor 3 (granulocyte), colony stimulating factor 2 (granulocyte-macrophage), granulocyte chemotactic protein 2, diubiquitin, ELAM-1, TNF More preferably, it comprises one or more of inducible protein 6, Exodus 1 and IL-1β.

  Alternatively, the method comprises determining a pattern of transcription product expression, wherein the transcript comprises at least some of the transcripts shown in Table 1 herein.

  Gene transcript or protein levels can be measured by any suitable means. When testing a large number of different gene transcripts, a convenient method is by hybridization (directly or after creation of cDNA or cRNA) of the sample to a gene chip array.

  When using gene chip technology, all of the genes (this term used herein includes the ESTs in Table 1) are present in chips commercially available from Affymetrix, and these chips are manufactured by the manufacturer. Can be used according to the protocol from In general, methods for the provision of microarrays and their use are described, for example, in WO84 / 01031, WO88 / 1058, WO89 / 01157, WO93 / 8472, WO95 / 18376 /, WO95 / 18377, WO95 / 24649 and EP- Also found in A-0373203, which can be referred to these documents and other documents in the art.

  When testing a large number of different proteins, a convenient method is antibody chip, multi-target ELISA or proteomic analysis. In general, methods for the provision of protein assays and their use can also be found, for example, in US Pat. No. 6,329,209 and US Pat. No. 2,656,508. Such methods are common in the art. More information on such methods is available by referring to http://www.marketresearch.com/map/prod/904611.html.

  Table 1 shows the names of the genes, which can be used to obtain their DNA sequences and encoded protein sequences from databases such as Genbank. Also, the individual sequences used on the Affymetrix chip we are using can be identified by the Affymetrix reference numbers shown in the table, which are publicly available and Genbank Can be directly associated with a registration number. In some cases, the EST gene sequence is also indicated by the Genbank accession number. A person skilled in the art can refer to either the Affymetrix reference number or the Genbank registration number in the practice of the present invention.

  Table 2 shows the cDNA sequence information for each of the probes used on the Affymetrix chip listed in Table 1. Table 1 lists the probe sets used to obtain the specific proteins / ESTs listed in this table, and Table 2 provides the cDNA sequence for each probe set listed in Table 1. ing.

  In Tables 2a and 2b, “n” represents in each case the region of the nucleic acid that was not probed by the probe set sequence on the gene chip. Therefore, to make the array as specific as possible, only selected regions of the complete cDNA are used, with the exclusion of some intervening sequences. Excluded sequences are those in which the single letter nucleotide code (a, g, c or t) is replaced by “n”.

Alternatively or in addition, quantitative PCR methods can be used. This is based on, for example, ABI TaqMan ^™ technology and is widely used in the art. It has been described in a number of prior art publications, see for example WO 00/05409. PCR methods require primer pairs that target the opposite strand of the target gene at an appropriate distance (typically 50-300 bases). Suitable target sequences for the primers can be determined by reference to the Genbank sequence described above.

The gene chip prior art provides a gene chip containing one or more of the genes of Tables 1a and 1b as part of a very large array, but a relatively small set of genes useful in the assessment of said symptoms Identification allows the provision of small chips (special gene arrays) specially designed to be suitable for use in the present invention.

  Thus, the present invention comprises at least five nucleic acids suitable for detection (directly or after production of cDNA or cRNA) of at least five transcripts shown in Table 1; A gene chip array is provided comprising a specific control; and optionally at least one control for the gene chip.

  In another embodiment of this aspect, the present invention is a gene chip array for the detection of at least 5 transcripts shown in Table 1 and detecting the presence of at least 5 transcripts Providing an array comprising at least 5 nucleic acids selected from Table 2; optionally a control specific for the transcript and optionally at least one control for the gene chip .

  The gene chip array comprises at least 5 different nucleic acids, preferably at least 10 or 20 different nucleic acids.

  Desirably, the number of nucleic acids suitable for detection of transcripts in Table 1 (or the number of sequences in Table 2) is n, and the number of control nucleic acids specific for individual transcripts is n ′ (where n 'Is 0-2n) and the number of control nucleic acids on the gene chip (eg, negative control, or “housekeeping” transcripts, abundant endothelial cell transcripts, relatively high levels in endothelial cells) If m is a control for detection of transcripts showing expression, transcripts showing a relatively high level of expression in the individual endothelium type being evaluated, where m is 0-100, preferably The number of sequences in the array is adjusted so that n + n ′ + m is at least 50%, preferably 75%, more preferably at least 90% of the nucleic acids on the chip.

As described above with respect to the protein chip gene chip, the provision of the transcripts in Table 1 and the proteins encoded thereby allows the production of suitable customized protein chips. Such a chip can be used in the form of an antibody chip or can include multi-target ELISA or proteomic analysis. In general, methods for the use of protein assays and their use can be found, for example, in US Pat. No. 6,329,209 and US Pat. No. 2,656,508. Such methods are common in the art.

  Thus, in yet another aspect, the present invention is a protein-based assay suitable for use in said method of the invention, wherein the protein is at least 5 proteins encoded by transcripts comprising those shown in Table 1. Optionally; a control specific for the protein; and optionally an assay for evaluation of at least one control for the assay.

  In a preferred embodiment, the protein-based assay is an antibody chip or ELISA, preferably a multi-target ELISA.

Assay Methods The assay methods of the present invention can be performed in a wide variety of formats, eg, against protein or nucleic acid components, or on whole cells in culture.

The invention, in its second aspect, is a method for assaying a modulator of a tissue inflammatory response or associated condition comprising:
a) providing a protein encoded by the transcript of Table 1;
b) contacting the protein with a candidate modulator of its activity; and c) determining whether the candidate modulator is capable of modulating the activity of the protein.

  Where the endothelial cell response to an inflammatory signal involves transcriptional down-regulation, the assay preferably relates to an activator of the protein, which assay preferably regulates the activity of the protein by the modulator. Including determining whether it can be increased. In this embodiment, the assay can be performed under conditions where the protein normally exhibits low activity or no activity.

  Where the endothelial cell response to an inflammatory signal is accompanied by transcript up-regulation, the assay is preferably directed to an inhibitor of the activity of the protein, and the assay is preferably such that the modulator is an activity of the protein. Determining whether it is possible to reduce In this embodiment, the assay can be performed under conditions where the protein is normally active.

  In this assay method, the measurement of modulation of activity depends on the nature of the protein being assayed. For example, a protein having an enzymatic function may be assayed in the presence of a substrate for the enzyme such that the presence of a modulator capable of modulating activity results in faster or slower substrate turnover. Is possible. The substrate can be a natural substrate of the enzyme or a synthetic analog. In either case, the substrate can be labeled with a detectable label to monitor its conversion to the final product.

  In the case of proteins with ligand binding function, such as receptors, candidate modulators can be tested for ligand binding function in a manner that results in antagonism or agonism of the ligand binding properties.

  In the case of proteins having DNA binding activity, such as transcriptional regulators, it is possible to measure DNA binding activity or transcriptional activation activity, where the modulator can increase or decrease such activity. For example, DNA binding can be measured in a mobility shift assay. Alternatively, a DNA region to which the protein binds is operably linked to a reporter gene (and, if necessary, a promoter region and / or a transcription initiation region between the DNA region and the reporter gene) to transcribe the gene. And that this transcriptional modulation can be observed when it occurs. Suitable reporter genes include, for example, chloramphenicol acetyltransferase, or more preferably a fluorescent reporter gene, such as green fluorescent protein.

Alternatively, or in a third aspect, the invention provides a method for assaying a modulator of a tissue inflammatory response or a related condition comprising:
a) providing cultured endothelial cells;
b) contacting the cell with a candidate modulator of the condition;
c) determining whether the candidate modulator is capable of modulating the level of at least one transcript selected from the transcripts of Table 1.

  In the first step of the method, the endothelial cells are cultured. Any suitable method can be used to collect and culture the cells. Techniques for culturing endothelial cells are common in the art.

  Candidate modulator compounds can be natural or synthetic compounds used in drug screening programs. It is also possible to use extracts of plants, microorganisms or other organisms that contain several characterized or uncharacterized components. Combinatorial library technology (including solid phase synthesis and parallel synthesis methods) provides an efficient way to test a potentially vast number of different substances for their ability to modulate interactions. Such libraries and their use are known in the art, especially for all types of natural products, small molecules and peptides. A number of such libraries are commercially available and are sold for drug screening programs of the type foreseen here by the present invention.

  Another class of candidate modulators includes antibodies or binding fragments thereof that bind to a protein target.

  Examples of antibody fragments capable of binding to an antigen or other binding partner include Fab fragments consisting of VL, VH, Cl and CH1 domains; Fd fragments consisting of VH and CH1 domains; Fv fragment consisting of VH domain; dAb fragment consisting of VH domain; isolated CDR region and F (ab ′) 2 fragment, bivalent fragment (including two Fab fragments linked by disulfide bridge in hinge region) It is done. Single chain Fv fragments are also included. Protein-specific antibodies are derived from recombinantly produced libraries of expressed immunoglobulin variable domains, for example, using lambda or filamentous bacteriophages that display functional immunoglobulin binding domains on the surface. It is possible to obtain. See, for example, WO 92/01047. Such techniques allow for the rapid generation of antibodies against antigens, which can then be screened according to the present invention.

  Another class of candidate molecules are peptides based on fragments of the protein sequence to be repressed. In particular, protein fragments corresponding to other proteins or portions of proteins that interact with DNA can be targets for small peptides that act as competitive inhibitors of protein function. Such peptides are, for example, 5 to 20 amino acids long.

  The peptides may also provide the basis for mimetic design. Such mimetics include the analysis of peptides to determine amino acid residues or parts of their side chains that are essential and important for biological activity to define a pharmacophore (pharmacophore), followed by an appropriate tertiary It may be based on modeling of the pharmacophore to design mimetics that retain the essential residues or parts thereof in an original relationship. There are various computer-aided technologies in the art to facilitate the design of such mimetics.

  Cell-based assay methods can be designed to measure gene expression at either the transcriptional or translational level. When measuring a transcription product, it is possible to measure this using the method of the first aspect of the present invention, for example, on a gene chip or by multiplex PCR.

  As noted above, if the transcript is one that is down-regulated in response to an inflammatory signal, the assay preferably regulates expression of the gene (eg, by increasing the amount of transcript). It relates to the substance to be increased. Such material can include the coding sequence of the gene itself (ie, it can be a gene therapy vector). Where the transcript is up-regulated in response to an inflammatory signal, the assay preferably relates to a substance that reduces the expression of the gene.

  Cell based assay methods can be used to screen candidate modulators as described above. They can also be used to screen other classes of candidate modulators, including antisense oligonucleotides. Such oligonucleotides are typically 12-25, such as about 15-20 nucleotides in length, and include or modify modified backbone structures, such as methylphosphonate and phosphorothioate backbones, to help stabilize the oligonucleotide. It is possible to become more. The antisense oligonucleotide can be derived from the coding region of the target gene or can be derived from a 5 'or 3' untranslated region. Candidate molecules can be further processed into siRNA by RNAi, a short double-stranded RNA molecule that is a sequence specific to a gene transcript, or by a cell, for example, can be supplied into a cell as a DNA sequence Longer RNA sequences (eliRNA or expressed long interfering RNA). They can also include ribozymes that specifically target transcript mRNA, ie, catalytic RNA molecules that cleave other RNA molecules of a particular nucleic acid sequence. General methods for the construction of ribozymes are known in the art.

  In a preferred embodiment of these aspects of the invention, the assay method described above is used to measure the level of transcript from Table 1b or the modulator of protein activity.

  The modulators obtained according to these aspects of the invention can be used in methods of modulating a tissue inflammatory response and / or symptoms associated therewith (eg, inflammatory symptoms) in a patient.

  Accordingly, the present invention also provides a pharmaceutical composition comprising a modulator of a tissue inflammatory response and / or symptoms associated therewith and a pharmaceutically acceptable carrier.

  Generally, the modulator is formulated with one or more pharmaceutically acceptable carriers suitable for the selected route of administration for the subject to be administered. In the case of a solid composition, as a conventional non-toxic solid carrier, for example, pharmaceutical grade mannitol, lactose, cellulose, cellulose derivative, starch, magnesium stearate, sodium saccharin, talc, glucose, sucrose, magnesium carbonate, etc. may be used. it can. A pharmaceutically administrable liquid composition is, for example, a solution or suspension in which a modulator and optionally a pharmaceutical adjuvant are dissolved or dispersed in a carrier (eg, water, saline, water-soluble dextrose, glycerol, ethanol, etc.). It can be prepared by forming a liquid. If desired, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like (eg sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, Ethanolamine oleate and the like). Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 15th Edition, 1975. The composition or formulation to be administered will, in each case, contain an amount of the active compound effective to alleviate the condition being treated.

  The route of administration may depend on the condition of the subject to be treated, but since endothelial cells form a vascular lining structure, administration into the bloodstream (eg, intravenous injection) is one possible route.

  The present invention also provides a modulator of tissue inflammatory response and / or associated symptoms for use in therapy.

  Furthermore, the present invention provides the use of a modulator of tissue inflammatory response in the manufacture of a medicament for the treatment of conditions associated with tissue inflammatory response.

Identification of a number of previously unknown ESTs and other genes related to the regulation of endothelial cells by vector inflammatory signals provides the basis for a novel vector system useful in the above and further aspects of the present invention. To do.

  Preferably, the sequence encoding the transcript listed in Table 1b is operably linked to a regulatory sequence capable of effecting expression of the coding sequence by the host cell. That is, the vector is an expression vector.

  The term “functionally linked” means that the components described are arranged in a relationship that allows them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

  Suitable host cells include bacteria, eukaryotic cells (eg, mammalian cells and yeast cells), and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, infant hamster kidney cells, COS cells and many other mammalian cell lines.

  The vector secretes a promoter or enhancer for expressing the inserted nucleic acid, a nucleic acid sequence that allows the polypeptide to be produced as a fusion, and / or a polypeptide produced in the host cell. As such, other sequences such as nucleic acids encoding secretion signals can be included.

  The vector may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of bacterial plasmids or a neomycin resistance gene in the case of mammalian vectors.

  The vector can further include enhancer sequences, terminator fragments, polyadenylation sequences and other sequences as appropriate.

  Vectors can be used in vitro, for example for the production of RNA, or can be used to transfect or transform host cells. The vector can also be adapted for use in vivo, for example in gene therapy methods. Systems for cloning and expression of polypeptides in a wide variety of host cells are well known. Vectors include vectors for gene therapy such as vectors based on adenovirus, adeno-associated virus, retrovirus (eg, HIV or MLV) or alphavirus vector.

  Promoters and other expression control signals can be selected so that the expression vector is compatible with the intended host cell. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. cerevisiae. Examples include the S. pombe nmt1 and adh promoters. Mammalian promoters include metallothionein promoters that can respond to heavy metals such as cadmium. It is also possible to use viral promoters, such as the SV40 large T antigen promoter or the adenoviral promoter. All these promoters are readily available in the art.

  Vectors for use in gene therapy for the production of the polypeptides encoded by the transcripts of Table 1b include vectors containing mini-gene sequences.

  Vectors can be transformed into the appropriate host cells to effect expression of the polypeptides of the invention. Accordingly, in yet another aspect, the present invention provides a method for preparing a polypeptide encoded by the transcript of Table 1b, wherein a host cell transformed or transfected with the expression vector is treated with the polypeptide. A method for the preparation is provided, comprising culturing under conditions that result in expression of the coding sequence by the vector and recovering the expressed polypeptide. Polypeptides can also be expressed by using in vitro systems such as reticulocyte lysates.

  A substantially isolated form of a polypeptide or fragment thereof encoded by the ESTs or genes of Table 1b constitutes another aspect of the invention. The polypeptide fragment is preferably at least 20 amino acids in size, preferably 25 amino acids to full length in size.

  Another aspect of the invention is a nucleic acid sequence encoding the polypeptide and fragments thereof. Such a nucleic acid sequence may be contained in a vector as described above, for example.

  For further details, see for example Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, Numerous known techniques and protocols for nucleic acid construct preparation, mutagenesis, sequencing, introduction of DNA into cells and manipulation of nucleic acids for gene expression and protein analysis are described in Current Protocols in Molecular Biology, Ausubel et al., John Wiley & Sons, 1992.

  If the gene of Table 1b or EST is present in the vector, it can be linked in frame with the translation initiation region for translation of the sequence, or it can be used for transcription of the antisense RNA. An antisense arrangement may be used.

TNFα, IL-1β and interleukin-8 regulate the amount of transcripts In order to enable the study of the effects of TNFα, IL-1β and interleukin-8 on the amount of transcript, seven independent HUVEC primary cultures were used. The cells were cultured for 24 hours with a mixture of 15 ng / mL each of TNFα, interleukin-1β, and interleukin-8. Subsequently, complex cRNA probes were prepared using RNA extracted from these cultures and hybridized to Affymetrix U95A gene chip (12,600 element Affymetrix gene array chip).

Analysis of Gene Chip Data The transcript amount data (“average difference”) was scaled overall so that the average gene expression of each chip (excluding control genes) was 1. In order to ensure that transcript expression levels were comparable between arrays, the “LOESS” function of the “R” statistical software system was converted to the log-transformed average difference value for each array in comparison to the control array. Applied. The selected control array had the closest similarity by Euclidean distance to the other six controls.

  The normalized transcript quantity data was then compared using the CyberT algorithm (version 7.03; sliding window = 301, Bayesian estimate = 21). This algorithm is an unpaired t-test modified by the inclusion of Bayes prior probabilities based on the variance of other transcripts in the dataset (Long et al., 2001, J. Biol Chem 276, 19937-19944).

  For further statistical analysis, the “R” statistical software system and GeneSpring Expression Analysis Software (Silicon Genetics, Redwood City, Calif.) Were used.

  Individually using the GO database (www.geneontology.org), Entrez (www.ncbi.nlm.nih.gov/entrez) or OMIM (www.ncbi.nlm.nih.gov:80/entrez/Omim) databases Transcripts were classified according to the function of the protein they encoded.

  In the table, the fold change is expressed as an unmatched average for 7 experiments (ie, average of treated inflammation / average of control).

To determine whether the differential transcriptome modulation at the transcript level in different endothelial cell types is comparable between different endothelial cell types, human coronary artery endothelial cells (HCAEC) and human uterine microvessels Another culture was obtained using each one of the endothelial cells (UtMVEC).

  Those three different cell types were treated with inflammatory signals as described above to assess the degree to which the amount of transcript was regulated in each of these three cell types. The pattern of regulation in these three cell types was also measured. It has been found that most transcripts are regulated in all three cell types, and some are regulated in only two or only one of these cell types.

  Transcripts that are regulated in all three cell types are considered particularly useful in the various methods of the invention. Because they appear to be significant in various inflammatory conditions affecting different organs.

  Furthermore, it has also been found that transcripts modulated by inflammatory signals in all three endothelial cell types tend to be strongly modulated by inflammatory signals in HUVEC.

Consensus data tables 1a and 1b show the consensus set of transcripts. These were selected based on their levels before and after treatment with inflammatory mediators differing by more than two orders of magnitude (in either direction) with a Bayesian P value of less than 0.001. These are classified according to gene family.

  Table 1 shows the degree of regulation in HUVEC cells and in HCAEC and UtMVEC cells.

EST
A number of previously unknown ESTs and genes identified in connection with the present invention are of particular interest as possible therapeutic agents for use in the various methods of the present invention and in therapy. These are summarized in Table 1b. The specific sequence of probes used to isolate these ESTs is shown in Table 2b.

Materials and methods:
Endothelial cell preparation HUVECs isolated from seven different individuals were cultured in microvascular endothelial cell growth medium 2 (EBM-MV, Biowhittaker). Proprietary mixture of human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, human fibroblast growth factor, human recombinant insulin-like growth factor, ascorbic acid, gentamicin, amphotericin B and FBS (5%) (Microvascular Bulletkit ^™ reagent, Biowhittaker ) Was added to the medium. At the fifth passage, each HUVEC isolate was split into ^two 175 cm ² culture flasks and grown to 70% confluence. For each HUVEC isolate, the growth media in one flask was replaced with fresh media containing a mixture of 15 ng IL-8, IL-1β and TNF-α, respectively. As a control, the medium in the replicate culture was replaced with fresh medium only. Cytokine-treated HUVECs and controls were further cultured for 24 hours.

  Human coronary EC and myometrial microtubule EC from one donor were purchased from Biowhittaker. Cells were cultured and treated under the same conditions as HUVEC.

RNA was extracted from each culture using RNA extraction Trizol reagent (Gibco / BRL, UK), then washed through an RNeasy ^™ spin column (Qiagen, UK) and further subjected to ethanol precipitation. An Alien 2100 bioanalyzer was used to assess that the RNA was intact.

Analysis of RNA Biotinylated target cRNA was prepared according to the Affymetrix protocol, hybridized to an Affymetrix human U95Av2 chip, and scaled globally using Microarray Suite Version 4.0 or 5.0 software.

Claims (33)

A method for assessing a tissue inflammatory response, comprising:
Making a quantitative measurement of the level of at least five transcripts shown in Table 1, or the protein encoded thereby, in a sample;
Comparing the amount of the transcript or protein thus measured to the level of the transcript obtained from a control sample.   2. The method of claim 1, wherein the sample comprises cells taken from a site suspected of being affected by a patient's tissue inflammatory response.   3. The method of claim 2, wherein the cells are endothelial cells or the sample is patient blood, serum or urine.   4. The method of claim 3, wherein the endothelial cells are human umbilical vein endothelial cells, human coronary artery endothelial cells or human uterine microvascular endothelial cells.   5. The method of any one of claims 1-4, wherein the control sample is taken from endothelial cells derived from tissue not affected by the patient's tissue inflammatory response.   5. The method according to any one of claims 1 to 4, wherein the control sample is taken at an earlier time point from tissue exhibiting an inflammatory response of the patient.   The tissue inflammatory response is: leukocyte migration across the endothelium, leukocyte migration to the inflamed tissue, leukocyte activation, endothelial cell activation and angiogenesis, and / or inhibition of apoptosis of leukocytes or endothelial cells 7. A method according to any one of claims 1 to 6, comprising one or more.   A method of diagnosing symptoms associated with a tissue inflammatory response, or a method of monitoring the progression of symptoms already associated with and diagnosed by a tissue inflammatory response, or monitoring the effectiveness of treatment of symptoms associated with a tissue inflammatory response The method of claim 1, which is a method.   The method according to claim 1, wherein the measurement is performed after one course of treatment of the patient.   The method according to claim 1, wherein the amount of at least 10 transcripts shown in Table 1 or the protein encoded thereby is measured.   11. The method of claim 10, wherein the amount of at least 20 transcripts shown in Table 1 or the protein encoded thereby is measured.   The method according to any one of claims 1 to 11, wherein the amount is measured by hybridization to a gene chip array.   The method according to any one of claims 1 to 11, wherein the amount is measured by quantitative PCR.   A method for the diagnosis of symptoms associated with a tissue inflammatory response, encoded by at least five of the transcripts of Table 1 in a sample derived from a patient suspected of being affected by such symptoms The above method comprising measuring the amount of endothelial cell-derived protein.   The tissue inflammatory response is associated with an inflammatory disease, vasculitis syndrome, atherosclerosis or related disease, chronic transplant rejection, or the condition is associated with tumor growth. The method described in 1.   The inflammatory disease is inflammatory bowel disorder, psoriasis, ischemia reperfusion, adult respiratory distress syndrome, asthma, allergic rhinitis, dermatitis, meningitis, encephalitis, uveitis, leukocyte emigration, central Nervous system inflammatory disorder, Alzheimer's disease, endometriosis, multiple sclerosis, multiple organ injury syndrome, alcoholic hepatitis, bacterial pneumonia, antigen-antibody complex mediated disease, lung inflammation (pleurisy, alveolitis, Including pneumonia, chronic bronchitis, bronchiectasis, cystic fibrosis and COPD), vasculitis, nodular polyarteritis, giant cell arteritis, microscopic polyarteritis, preeclampsia and autoimmunity 16. A method according to claim 15, comprising a disease.   17. A method according to any one of the preceding claims, wherein the comparison measures a change in transcript level or transcript quantity pattern between the sample and the control sample. The level / quantity to be measured is as follows:
Colony stimulating factor 3 (granulocyte), colony stimulating factor 2 (granulocyte-macrophage), granulocyte chemotactic protein 2, diubiquitin, ELAM-1, TNF inducible protein 6, Exodus1, IL-1β, VCAM-1 ICAM-1, IAP1, TNF inducible A20, RIPK2, MMP 10, TRAF1, JAK binding protein, bispecific phosphatase 4, IL-6, IL-8, Gro-γ, MCP-1, Gro-β, Gro-α, ENA-78, fractalkine, low molecular weight inducible cytokine subfamily A14, low molecular weight inducible cytokine A5, LI7 receptor, toll / interleukin 1 receptor-like 4, CCAAT, MAD-3, COX-2 , Mn SOD, NO synthase, L-kynurenine hydrolase, tissue factor Pathway inhibitor 2, laminin γ2, Toll-like receptor 2, natural killer cell transcript 4, TNFα-inducible protein 2, cationic amino acid transporter 2A, fatty acid binding protein 4, TNF receptor superfamily member 11b and TNF superfamily member 9
18. The method according to any one of claims 1 to 17, wherein the method is for at least one transcript or protein encoded thereby selected. Said transcript or protein encoded thereby is:
Colony stimulating factor 3 (granulocyte), colony stimulating factor 2 (granulocyte-macrophage), granulocyte chemotactic protein 2, diubiquitin, ELAM-1, TNF inducible protein 6, Exodus 1 and IL-1β
19. The method of claim 18, wherein the method is more selected.   A gene chip array suitable for use in the method according to any one of claims 1 to 19, comprising at least five nucleic acids suitable for detection of at least five transcripts shown in Table 1. Said array comprising optionally a control specific for said transcript; and optionally at least one control for said gene chip.   A gene chip array for the detection of at least five transcripts shown in Table 1, wherein at least five selected from Table 2 capable of detecting the presence of at least five transcripts The array comprising a nucleic acid; optionally a control specific for the transcript and optionally at least one control for the gene chip.   20. A protein-based assay suitable for use in the method of any one of claims 1-19, wherein at least five proteins encoded by transcripts comprising those shown in Table 1, and Said assay for the evaluation of a control specific for said protein and optionally at least one control for said assay.   23. The assay of claim 22, which is an ELISA or antibody chip. An assay method for determining a modulator of a tissue inflammatory response or associated symptoms, comprising the following steps:
a) providing a protein encoded by the transcript from Table 1;
b) contacting the protein with a candidate modulator of its activity;
c) The above assay method comprising determining whether the candidate modulator is capable of modulating the activity of the protein.   25. The assay method of claim 24, wherein the candidate modulator is an antibody or binding fragment thereof that binds to the protein.   25. The method of claim 24, wherein the candidate modulator is a fragment of the protein or a mimetic thereof. A method for assaying a modulator of a tissue inflammatory response or associated symptoms comprising the following steps:
a) preparing cultured endothelial cells;
b) contacting the cell with a candidate modulator of the condition;
c) The above assay method comprising determining whether said candidate modulator is capable of modulating the level of at least one transcript of Table 1.   28. The assay method of claim 27, wherein the candidate modulator is an antisense oligonucleotide or RNAi.   28. The assay method of claim 27, wherein the candidate modulator is a sense oligonucleotide.   30. Use of a modulator obtained according to the assay method of any one of claims 24 to 29 for the treatment of a tissue inflammatory response or a related condition.   A vector comprising a sequence encoding the transcript from Table 1b operably linked to a promoter for transcription of the sequence.   32. The vector of claim 31, wherein the sequence is linked in frame with a translation initiation region for translation of the sequence.   32. The vector of claim 31, wherein the sequence is in an antisense configuration.