JP2007536939A - Immune cell biosensor and method of use thereof - Google Patents

Abstract

The present invention relates to immune cells useful in detecting changes in physiological conditions that provide a method of diagnosing disease or monitoring the course of treatment of a patient. Also provided are arrays of antigen presenting cell-specific markers for detecting changes in physiological conditions and methods for detecting such changes.

Description

The present invention relates to immune cells useful in detecting changes in physiological conditions that provide a method of diagnosing disease or monitoring the course of treatment of a patient.

Background In many diseases such as cancer, autoimmune diseases or cardiovascular diseases, peptides of normal or abnormal proteins that cannot be found on the cell surface of healthy individuals are presented on the cell surface. Insufficient antigen presentation in humans prevents the human immune system from controlling and eliminating numerous pathogen infections and malignant cell proliferation. The success of therapeutic vaccines and immunotherapy for chronic infections relies on the development of new methods of antigen presentation that are efficient in inducing various immune responses that can control and eliminate protective antigens.

  The ability of T cells to recognize an antigen depends on the association of the antigen with MHC class I (MHC-I) or class II (MHC-II) proteins. For example, cytotoxic T cells respond to antigen in association with MHC-I protein. Thus, T cells that kill virus-infected cells will not kill cells infected with this virus, even if the cells do not express the appropriate MHC-I protein. Helper T cells recognize the MHC-II protein. Helper T cell activity generally depends on both antigen recognition in antigen presenting cells and the presence of “self” MHC-II protein in these cells. This requirement of recognizing an antigen in relation to a self MHC protein is called MHC restriction. MHC-I protein is found on the surface of virtually all nucleated cells. MHC-II protein is found in cells including splenic macrophages, B cells and dendritic cells (DC) and skin Langerhans cells.

  A critical step in initiating an immune response in mammals is activation of CD4 + helper T cells that recognize major histocompatibility complex (MHC) -II restricted foreign antigens. These antigens are captured and processed in the cellular endosomal pathway of antigen-presenting cells such as dendritic cells. In endosomes and lysosomes, the antigen is processed into small antigen peptides and presented on the Golgi fraction of MHC-II to form the antigen-MHC-II complex. This complex is expressed on the cell surface and expression induces activation of CD4 + T cells.

  Another significant event in the induction of an effective immune response in animals involves CD8 + T cell and B cell activation. CD8 + cells are activated when they pass through the cells in a manner such that the desired protein is presented on the cell surface as a processed protein to form a complex with the MHC-I antigen. B cells can interact with antigen by surface immunoglobulins (IgM and IgD) without the need for MHC proteins. However, activation of CD4 + T cells stimulates all arms of the immune system. As a result of activation, CD4 + T cells (helper T cells) produce interleukins. These interleukins help activate other arms of the immune system. For example, helper T cells are interleukin-4 (IL-4) and interleukin-5 (IL-5) that help B cells produce antibodies; interleukin − that activates CD4 + and CD8 + T cells. 2 (IL-2); as well as γ-interferon that activates macrophages. Helper T cells that recognize MHC-II-restricted antigens play a central role in the activation and clonal expansion of cytotoxic T cells, macrophages, natural killer cells, and B cells, so helpers in response to antigens The initial event of activating T cells is critical for the induction of an effective immune response against that antigen.

  Peptides and proteins expressed in diseased cells can be used as markers for the identification of such abnormal cells. Furthermore, detection of antibodies against these peptides in serum or other biological fluids can also be used as an indicator of risk or as a prognostic indicator. However, the concentrations of these disease-related peptides are very low, and their isolation and identification are usually only effective when the disease has prevailed in an individual, by which normally effective treatment is not possible. Become. There remains a need in the art for a rapid and sensitive assay for detecting mammalian morbidity.

SUMMARY OF THE INVENTION The present invention is based on the flexibility of antigen presenting cells and the specific metabolic changes that APCs, particularly DCs, undergo after encountering an antigen. These changes can be quantified and provide information regarding the immune status and microenvironment of the mammal from which they were obtained when compared to APC reference positive (antigen contact) and negative (untreated) controls.

  In one aspect, the invention provides a step of obtaining a blood sample having a subpopulation of antigen-presenting cells from a mammalian subject, substantially isolating the antigen-presenting cells from the blood sample, Inducing a genomic or proteomic mammalian subject signature, wherein the mammalian subject signature indicates a metabolic state of the subject's antigen presenting cells, one of the antigen presenting cells from a reference subject having a disease state, or Deriving a plurality of genomic or proteomic reference signatures, and comparing the signature of the mammalian subject with the reference signature, wherein the match between the signature of the mammalian subject and the reference signature is indicative of a disease state in the mammal A diagnostic method is provided that includes a step of indicating presence. The mammalian subject is preferably a human but may also be an animal subject such as a dog, cat, horse, pig, sheep, goat or other animal. In one embodiment, the antigen presenting cell is a dendritic cell. In another embodiment, the disease state is cancer or a cell proliferative disorder. In yet another embodiment, the disease state is a pathogen infection. In yet another embodiment, the pathogen infection is a viral infection. In yet another embodiment, the pathogen infection is a bacterial infection.

  In another aspect, the present invention provides an array including a plurality of addresses, each nucleic acid sample corresponding to a gene expressed by an antigen-presenting cell being added to each address. In one embodiment, the array further comprises a plurality of secondary addresses to which each secondary address is appended with a nucleic acid sample corresponding to a gene expressed by an antigen presenting cell that has experienced the antigen. In another embodiment, the antigen presenting cell is a dendritic cell. In yet another embodiment, the antigen is a cancer antigen. In yet another embodiment, the antigen is a viral antigen. In yet another embodiment, the antigen is a bacterial antigen. In yet another embodiment, the antigen is a fungal antigen.

  In yet another aspect, the present invention provides an isolated antigen-presenting cell population, culturing antigen-presenting cells in the presence of a food-borne pathogen, thereby providing a proteomic or genome specific for the food-borne pathogen Creating a reference cell having a reference signature, obtaining a reference signature, obtaining a food sample, culturing naive antigen-presenting cells with the sample food, obtaining a sample signature from the cultured antigen-presenting cells; And comparing a sample signature with a reference signature, wherein the match between the sample signature and the reference signature indicates the presence of a foodborne pathogen in the food. In one embodiment, the antigen presenting cell is a dendritic cell. In another embodiment, the foodborne pathogen is a bacterial pathogen. In yet another embodiment, the foodborne pathogen is a viral pathogen. In yet another embodiment, the foodborne pathogen is a prion pathogen. In a related aspect, the present invention assayes for obtaining antigen-presenting cells from livestock mammals, and APC contact with food-borne pathogens in livestock mammals, and changes in gene and protein expression in APC resulting from antigen contact. Provide stages. In one embodiment, the antigen presenting cell is a dendritic cell. In another embodiment, the foodborne pathogen is a bacterial pathogen. In yet another embodiment, the foodborne pathogen is a viral pathogen. In yet another embodiment, the foodborne pathogen is a prion pathogen, such as a prion that causes bovine spongiform encephalopathy (BSE).

  In yet another aspect, the invention provides obtaining a blood sample having a subpopulation of antigen presenting cells from a patient undergoing treatment for a disease, substantially isolating antigen presenting cells from the blood sample, Inducing genomic or proteomic patient signatures for isolated antigen presenting cells, wherein the patient signature indicates the metabolic state of the subject's antigen presenting cells, antigen presenting cells from a reference subject having the same disease as the patient Deriving one or more genomic or proteomic reference signatures of the patient and comparing the patient signature with the reference signature, thereby reducing the consistency of the patient signature with the reference signature during treatment, thereby reducing the disease A diagnostic method is provided comprising the step of showing the effectiveness of the treatment in therapy. In one embodiment, the disease is a cell proliferative disease or cancer and the treatment is administration of an antineoplastic agent. In another embodiment, the disease is a cell proliferative disease or cancer and the treatment elicits an immune response against the cell proliferative disease. In yet another embodiment, the disease is an autoimmune disease and the treatment reduces the autoimmune response. In yet another embodiment, the disease is a bacterial infection and the treatment is administration of an antibacterial agent. In yet another embodiment, the disease is a viral infection and the treatment is administration of an antiviral agent. In yet another embodiment, the disease is a fungal infection and the treatment is administration of an antifungal agent. In yet another embodiment, the disease is a genetic disease and the treatment is gene replacement therapy.

  In another aspect, the present invention provides an antigen-presenting cell cultured in the presence of an antigen, wherein the antigen expresses a plurality of genes that are specifically upregulated in response to an antigen load. I will provide a. In one embodiment, the antigen presenting cell is a dendritic cell. In yet another embodiment, the antigen is a cancer antigen. In yet another embodiment, the antigen is a viral antigen. In yet another embodiment, the antigen is a bacterial antigen. In yet another embodiment, the antigen is a fungal antigen. In yet another embodiment, the antigen is a prion antigen. In one aspect, a specific polypeptide (marker protein) produced in response to antigen contact is isolated. These are used to form antibodies, and the antibodies are used in subsequent assays involving APCs isolated from patients, whereby the polypeptides expressed in the patients from which APCs are isolated are converted to immunological assays, For example, qualitatively and quantitatively identified by ELISA, FAC, RIA and similar techniques.

  In another aspect, the present invention provides a step of determining the proteomic signature of an antigen presenting cell contacted with an antigen. In one embodiment, the proteomic signature is obtained by subjecting antigen presenting cells to SELDI mass spectrometry. In another aspect, proteomic signatures are obtained by subjecting antigen presenting cells to MALDI-O-TOF and other forms of mass spectrometry. In yet another aspect, the present invention provides a proteomic signature obtained from an antigen presenting cell contacted with an antigen. In one embodiment, the antigen presenting cell is a dendritic cell. In yet another embodiment, the antigen is a cancer antigen. In yet another embodiment, the antigen is a viral antigen. In yet another embodiment, the antigen is a bacterial antigen. In yet another embodiment, the antigen is a fungal antigen. In yet another embodiment, the antigen is a prion antigen.

  In one aspect, the invention provides obtaining a blood sample having a subpopulation of antigen-presenting cells from a diseased mammal; substantially isolating antigen-presenting cells from the blood sample, one from the antigen-presenting cells. Or deriving a plurality of marker polypeptides, wherein the marker polypeptides are expressed in antigen presenting cells in response to antigen contact, and the contacted antigen is associated with or is a causative agent of the disease; Obtaining an antibody against the marker polypeptide; and detecting the presence or absence of the polypeptide binding to the antibody in the subject's antigen-presenting cells, wherein the presence of the polypeptide confirms the presence of the disease in the subject. A diagnostic method is provided. Detection of polypeptide binding to the antibody can be performed using assays such as ELISA, RIA, FRET, FAC and other immunodetection methods. In one embodiment, the antigen presenting cell is a dendritic cell. In one embodiment, the disease is cancer or a cell proliferative disease. In one aspect, the disease is a pathogen infection. In one embodiment, the disease is a viral infection. In one aspect, the disease is a bacterial infection. In one aspect, the disease is a prion infection. In one aspect, the disease is a fungal infection.

  In another aspect, the present invention includes a method of diagnosing contact with an antigen, comprising detecting a protein / gene expression level present in a sample of mammalian tissue or mammalian biological fluid that has not contacted the antigen. . Subsequently, the protein / gene expression level present in the sample of mammalian tissue or mammalian biological fluid in contact with the antigen is detected. Determine the difference in the detected amount of protein / gene expression between the contacted and uncontacted samples. Compare the difference with the expected protein / gene expression library for a given antigen. Finally, it is assessed whether the difference indicates contact with a specific antigen. The present invention is particularly useful because it can provide a diagnosis of whether a person has come into contact with an antigen prior to the onslaught of any symptoms. The invention also includes detecting a gene expression / protein pattern present in a mammalian tissue or mammalian biological fluid sample that may have been contacted with an antigen, expressed in those tissues of an individual that may have been contacted Determining a relative amount of gene or protein panel expression for a given housekeeping gene and protein, comparing a relative amount difference for an expected gene expression / protein library for a given antigen; and known Assessing whether the difference indicates that contact has occurred with the listed virulence factors, antigens previously mixed with unknown or potentiating agents, and The present invention relates to a method for diagnosing contact with a person. Housekeeping genes are genes that tend not to change when in contact with an antigen.

  These aspects and other features will become apparent from the discussion below.

DETAILED DESCRIPTION The present invention addresses the observed flexibility of antigen presenting cells (APCs) and the specific changes in gene and protein expression that occur in human dendritic cells and monocytes in response to pathogens, tumors and harmful pathogens. Based on their use for rapid detection. Antigen presenting cells, particularly dendritic cells (DC) macrophages and monocytes (M) and to a lesser extent, B cells, are constantly sampling the various microenvironments found in mammalian organisms. For example, DC cells are found immature in most tissues (CD1a +, CD83 ^Low ), which recognize and phagocytose pathogens and other antigens (Poindexter, et al., (2004) Breast Cancer Res. 6 (4): See 408-415). Platelets are not themselves cells, but platelets can also bind to infectious microorganisms and serum proteins, be phagocytosed, and be considered a reservoir for detecting pathogens and cell fragments such as tumor cells and apoptotic cell debris (See Youssefian, et al., Host defense role of platelets: engulfment of HIV and Staphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation. Blood. 2002 Jun 1; 99 (11): 4021-9) . Thus, antigen presenting cells generally refer to cells and cell fragments that have the ability to internalize antigens and present these antigens to other cells. Exemplary antigen presenting cells include lymphoid cells such as T cells, B cells, lymphoid-related dendritic cells and natural killer cells, and bone marrow lineage cells such as bone marrow-related dendritic cells, macrophages, monocytes, megakaryocytes Including, but not limited to, myeloid cells such as platelets, granulocytes and neutrophils. Strongly phagocytic cells such as macrophages, monocytes and dendritic cells are preferred.

  By direct contact with antigens or other pathogenic factors, these antigen-presenting cells mature and increase antigen presentation, expression of costimulatory molecules, expression of cytokines and subsequent untreated T cells and dendrites in lymphoid organs Characterized by stimulation of other cell specific markers such as surface CD83 expression in cells. This maturation process is regulated by a number of corresponding changes in gene expression in these cells that can be measured qualitatively and quantitatively. The resulting series of gene expression changes is highly specific and responds specifically to the specific antigen contacted by the APC. APC, for example, can identify specific peptides, glycopeptides, glycolipids and initiate similar but not identical responses when contacted with various antigens. Certain changes in APC gene and protein expression in response to antigen addition indicate a very specific measurable biological signature, indicating that APC has experienced the antigen and the nature of the antigen itself, e.g., its chemical composition And can be used to identify the origin. In certain embodiments, isolation of APC allows subsequent phagocytosed antigens or possibly whole pathogens to be extracted from APC and isolated, which can be further characterized by MS or similar tools. For example, DCs are known to internalize without killing viruses and bacterial pathogens (Sundquist et al., 2004, and Jantsch et al., 2003). Accordingly, one object of the present invention includes methods for recovering part and / or whole of a pathogen or antigen in addition to obtaining the genome / proteomic signature of the pathogen or antigen. Another object of the invention is the isolation of APC polypeptides that are upregulated in response to antigen contact. These polypeptides are highly specific markers for antigen contact, and their expression indicates that APC has experience with specific antigens. By isolating these polypeptides in whole or in part, antibodies can be formed and used in subsequent assays to determine antigen contact.

  For example, U.S. Pat.No. 6,316,197 to Das et al., Method of diagnosing of exposure to toxic agents by measuring, by measuring distinct patterns of expression levels of specific genes. See distinct pattern in the levels of expression of specific genes). Contact of immature DC with LPS stimulation contributes to terminal differentiation into CD70 (+) DC (Iwamoto S., et al., Lipopolysaccharide stimulation converts vigorously washed dendritic cells (DCs) to nonexhausted DCs expressing CD70 and evoking long- lasting type 1 T cell responses., J Leukoc Biol. 2005 Apr 27; [Epub ahead of print])). Kumar, A., Kurl, RN Kryworuchko, M., Diaz-Mitoma, F., & Sharma, S. 1995. Detailed description of the relationship between EBV transformation and increased expression of c-myc and polyA polymerase (PAP) Differential effect of heat shock on RNA metabolism in human Burkitt's lymphoma B-cell lines. Leuk. Res, 19 (11) : See also 831-840. Hwang SL., Et al., Indoleamine 2,3-dioxygenase (IDO), describes upregulation of IDO in response to LPS or TNF-α stimulation, activates dendritic cells and chemotaxis to chemokines Indoleamine 2,3-dioxygenase (IDO) is essential for dendritic cell activation and chemotactic responsiveness to chemokines), Cell Res. 2005 Mar; 15 (3): 167-75; and human umbilical cord blood monocytes Originated DCs kill blood tumor cells by activation with lipopolysaccharide (LPS) or γ-interferon (IFN-γ), which is associated with increased TNF-α-related apoptosis-inducing ricand (TRAIL) expression in cord blood DC cytoplasm See also Shi J., et al., Cancer Sci. 2005 Feb; 96 (2): 127-33, which describes acquiring the ability to Human herpesvirus-6 (HHV-6) dramatically suppressed the secretion of interleukin-12 (IL-12) p70 by DC, while the closely related beta herpesvirus HHV-7 did not. Smith, AP, et al., J Virol. 2005 Mar, detailing that other cytokines affecting maturation, i.e., IL-10 and tumor necrosis factor alpha production were not significantly altered. 79 (5): See also 2807-13. Komer et al. Described upregulation of 103 genes in macrophages that are upregulated in response to Bacillus anthracis lethal toxin (LeTx). Similarly, Tucker et al., Describe LeTx cleavage of mitogen-activated protein kinase kinase (MAPKK) in a variety of different APC cell types. Expression of genes regulated by MAPKK activity did not change significantly, but a series of genes under the control of glycogen synthase kinase-3-β (GSK-3β) altered expression after LeTx treatment. Nitric oxide by APC in response to contact with Leishmania major, wild boar disease (Francisella tularensis), Mycobacterium bovis (BCG) and Plasmodium berghei Green, SJ, Scheller, LF, Marletta, MA, Seguin, MC, Koltz, FW, Slayter, M., Nelson, BJ, & Nacy, CA 1994. Nitric oxide describes the regulation of (NO) production See also: Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens. Immunol. Lett, 43 (1-2): 87-94. Hernychova, L., Kovarova, H., Macela, A., Kroca, M., Krocova, Z. & Stulik, J. 1997. Early results of macrophage wild-type fungus interactions under the influence of different genetic backgrounds in mice consequences of macrophage-Francisella tularensis) interaction under the influence of different genetic background in mice). Immunol.Lett, 57 (1-3): 75-81 and Clemens, DL, Lee, BY & Horwitz, MA 2004. Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages. See also Infect. Immun., 72 (6): 3204-3217. Ng, LC, Forslund, O., Koh, S., Kuoppa, K. & Sjostedt, detailing a total of 22 different genes that are up-regulated in response to Y. pestis infection A. 2003. The response of murine macrophages to infection with Yersinia pestis as revealed by DNA microarray analysis. Adv. Exp. Med Biol, See also 529: 155-160. These genes include unknown ESTs, cytokines, cytokine enzymes, receptors, ligands, transcription factors, transcription factor inhibitors, proteins involved in the cytoskeleton, and factors known to be associated with the cell cycle and proliferation There are seven genes that code for, three of which play a role in apoptosis. LPS treatment of APC details down-regulating the expression of transcription factors, proto-oncogenes, apoptosis-related proteins (cysteine proteases), intracellular kinases and growth factors, Saban, MR, Hellmich, H., Nguyen , NB, Winston, J., Hammond, TG & Saban, R. 2001. Time course of LPS-induced gene expression in a mouse model of genitourinary inflammation. See also Genomics, 5 (3): 147-160. Gene upregulation in response to LPS is interleukin-6 (IL-6) receptor, α- and β-nerve growth factor (α- and β-NGF), vascular endothelial growth factor receptor-1 (VEGF R1) , Observed in a cluster containing CC chemokine receptor and P-selectin. Another close cluster of prominent expression included proto-oncogenes c-Fos, Fos-B, Fra-2, Jun-B, Jun-D and Egr-1. Almost all interleukin genes were up-regulated 1 hour after LPS stimulation. Nuclear factor κB (NF-κB) pathway genes assembled in a single cluster with peak expression 4 hours after LPS stimulation. On the other hand, most of the interleukin receptors and chemokine receptors showed late expression peaks 24 hours after LPS contact. Mendis, C., Das, R., Hammamieh, R., Royaee, A., Yang, D., Peel, S. & Jett, M. 2005. Transcriptional response signature for staphylococcal enterotoxin B See also Genes Immun., 6 (2): 84-94. of human lymphoid cells to staphylococcal enterotoxin B).

  Thus, these polypeptides and other APC polypeptides provide protein markers that indicate antigen contact. In one aspect, these polypeptide markers are isolated and used to form antibodies. Anti-APC marker antibodies are useful in assays that can be used to detect expression of APC marker polypeptides in cells obtained from patients suspected of having antigenic contact. In one embodiment, the anti-APC marker antibody is an immunological detection method such as fluorescence activated cell sorting (FACS), fluorescence resonance emission tomography (FRET), radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Used in assays that use Other immunological detection assays are known to those skilled in the art and are suitable for the detection methods described herein.

  Thus, changes in APC in response to antigen addition can be used to assay for prodromal (non-disease) individuals and monitor the progression of the disease or the effectiveness of the therapy in treating the disease. Can do. For example, the effect of the antibiotic linezolid on the secretion of exotoxins by Staphylococcus aureus, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blot Bernardo, K., Pakulat, N., Fleer, S., Schnaith, A., Utermohlen, O., Krut, O., Muller, S. & Kronke, which describes the analysis by a combination of analysis , M. 2004. Subinhibitory concentrations of linezolid reduce Staphylococcus aureus virulence factor expression. Antimicrob. Agents Chemother., 48 (2): 546-444 . Similarly, changes in APC in response to antigen addition can be used to assay individuals who have been exposed to biological weapons, during early diagnosis of high-risk contact individuals; and at an early stage of developing symptoms Can be used to monitor a person. For example, changes in APC in response to antigen addition from viral or bacterial pathogens can quickly identify these pathogens and determine hours or days earlier than the appearance of the final pathogen in plasma. In a related application, the present invention can be used to monitor the effectiveness of vaccination by assaying the interaction of DC with one or more components of the vaccine. Similarly, changes in APC in response to antigen addition from the tumor allow the detection of the tumor before the individual develops symptoms, thereby allowing early aggressive treatment. In addition, changes in APC in response to contact with industrial chemicals or biological warfare weapons can identify unknown causal factors that individuals may be in contact with.

  APC works as a natural immune biosensor in the body. These cell types circulate through the whole body tissue and are responsible for most if not all of the body tissue by sampling the microenvironment. In conducting the survey, they look for areas of tissue where there are danger signals, ie increased mitotic activity or viral / bacterial infection (Crawford et al., 2003). When this signal is detected, APC initiates early transcriptional changes, cell surface antigens are expressed, and inflammatory mediators are released (Crawford et al., 2003). These cellular alterations are required for the recruitment of other inflammatory cells to the site of involvement and improved cell-to-cell contact. Antigen-presenting cells such as DC have pattern recognition receptors, which can bind to and distinguish various pathogens (Chaussabel et al., 2003). Other receptors include Toll-like receptors, ICAMs such as ICAM-1, DCSIGN, and others.

  As mentioned above, APCs create their own gene signatures in response to contact with various pathogens. Disordered gene expression in DCs and macrophages infected with bacteria, Candida, influenza or different parasites was examined using oligonucleotide arrays. Of the approximately 6800 gene samples, about 1300 genes It is suggested to prove a large change in the expression pattern after contact (see Huang et al., The plasticity of dendritic cell responses to pathogens and their components, Science, 294: 2001). For example, DC expresses a C-type lectin as a pathogen recognition receptor, such as DC-specific ICAM-3 Grabbing nonintegrin (SIGN) / CD209, as a DC HIV-1 receptor as well as Mycobacterium tuberculosis (Mycobacterium tuberculosis), Helicobacter pylori, Leishmania mexicana, Schistosoma mansoni, and other pathogen surface glycans have been identified (Appelmelk et al. (2003), J Immunol., 170: (4) 1635-9). See also Hofer et al., (2001) Immunol. Rev., Jun: 181: 5-19 and Puldendran et al., (2001), J. Immunol. Nov.:167(9): 5067-76. These receptors and the cellular pathways with which they interact provide unique markers for monitoring DC activation and response. More specifically, changes in DC response are measurable, providing pathogen-specific signatures that demonstrate the interaction of specific disease-causing factors with DC. Several MMP genes describe in detail that transcription is active in cells tested after contact with various stimuli such as phorbol ester, lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) , Machein, U. & Conca, W. 1997. Expression of several matrix metalloproteinase genes in human monocytic cells. Adv. Exp. Med. Biol, 421: 247- See 251.

  Harmful environmental factors can trigger APC-specific immune cell responses themselves, or destroy cells and tissues, increase inflammation, extravasation and APC activation in response to cytokines and various cellular factors Harmful environmental factors can also be detected by the methods described herein. These characteristics of human APC make it suitable for the rapid detection of contact with any virulence factor, such as infectious agents, tumors, toxins or toxic industrial chemicals (TIC) or weapons of mass destruction (WMD) It becomes.

  The described APCs, preferably monocytes and most preferably DCs, are useful for detecting changes in a subject's physiology, particularly in response to diseases such as infectious diseases and cancer. As used herein, the term antigen is generally foreign to a healthy mammal, or is natural to a mammal, but is mutated, abnormal or at a concentration in a mammal in a pathological state Is used broadly to refer to any composition in which an increase is seen. Thus, antigens include whole pathogens such as bacteria, viruses, fungi, protozoa as well as one or more components of pathogens, for example, bacterial antigens include lipopolysaccharide (LPS), and viral antigens include HIV gp120 or influenza Including viral coat proteins such as hemagglutinin, fungal antigens include the cell wall derived protein mannin. Prions are also antigenic and exhibit specific peptide sequences associated with disease states. Antigens also include tumor-related proteins and peptides such as carcinoembryonic antigen (CEA) and aberrantly glycosylated mucins (MUC) and numerous other tumor-specific antigens and proteins such as bcl-2, survivin, hepsin Including. Thus, common features of antigens or antigenic factors result in specific biochemical changes in APCs, such as upregulation of antigen presenting proteins and co-receptors, and APC maturation and proliferation, tissue migration and antigens It is an effect on APC in that it produces other characteristics that indicate contact. A detectable increase in APC is formed or activated in a mammal that has been contacted with an antigen by more than a two-fold increase in the number of APCs compared to the level of APC in an uncontacted or healthy mammal, or An increase induced to mature. Assay techniques for measuring DC and other blood cells are well known in the art, but are not limited to FACS using, for example, the mature DC cell markers CD2 + and CD83 + or the immature marker CD1a +.

  In one aspect, the present invention provides an immune cell based method for monitoring a patient's response to a disease state. Measuring pathogen or tumor-specific genomes and proteomic expression patterns or signatures provides an improved way to continuously monitor a patient's immune response to a disease state. Use this information in conjunction with other relevant medical information, such as a reduction in the mass of tumor patients or systemic tumor tissue, or a reduction in viral load in HIV-infected patients, or elimination of actinomycetes in tuberculosis patients Allowing the monitoring of therapeutic effects in response to eg administration of chemotherapy, antiviral therapy or antibiotics.

  Thus, APC provides a useful diagnostic tool for identifying antigens and monitoring an individual's health based on changes in cellular metabolism. Measurable changes occur in the expression of secretory factors such as numerous genes, proteins and cytokines, and antigens can also be detected in the cytoplasm of APCs (such as in the cytoplasm of platelets). Thus, in one aspect, the invention provides an array of APC gene signatures, preferably an array of monocytes or DC signatures. The array includes oligonucleotides, oligoribonucleotides or multiple APC marker genes and protein polypeptides, ie, genes or protein products that are differentially expressed in antigen presenting cells. More preferably, the array comprises about 500-1000 specific markers at individual addresses of the matrix. Even more preferably, the array comprises about 5,000, about 10,000, about 20,000 or more genes or gene products shown on the array. Most preferably, the array is a genome wide array, such as a mature DC cDNA array. Affymetrix and Illumina (both systems are complementary) arrays are exemplary. Individual genes or gene products may be duplicated on the array, for example, as a control or for quantitative analysis of gene expression. The manufacture and use of such arrays are described in US Pat. Nos. 6,741,344, 6,733,977 and 6,733,964. A method and apparatus for selectively applying a material to a selected region or address of a substrate, eg, for the synthesis of an array of oligonucleotides, to the substrate is further described in US Pat. No. 6,667,394. The gene array produced is very representative of the host response to pathogens (typically over 52,000 genes), for example one or several genes as in the case of identification by Q-PCR. It does not depend on identification (which is essentially biased). Proteomic data can be generated by various techniques, but is not limited to the use of, for example, surface plasmon resonance or mass spectrometry (such as MALDI or SELDI). The combination of information obtained using genomic and proteomic methods in a high-throughput screen format such as a DC gene array provides exceptionally specific diagnostic data, thus providing a powerful tool for antigen identification or patient monitoring.

  In order to form a diagnostic array based on APC for various purposes, in order to obtain a data set on how APC, in particular DC, reacts as a result of contact with different antigens, a number of Kinds of arrays are made. The use of a particular array depends on the chemical composition and varies depending on whether the array has other chemical moieties, such as nucleic acids, peptides, both, or lectins. As a general illustration, arrays such as GeneChip® from Affymetrix use biotin-labeled cRNA made from cell extracts. About 5 micrograms of total RNA is a suitable starting material. The cRNA made from the RNA sample is contacted with the array and hybridized to an appropriate target. The array is then washed and stained with, for example, streptavidin phycoerythrin, and then visualized using an Affymetrix GeneChip® Scanner 3000 or Agilent GeneArray® Scanner. This technique, as well as other known methods using immunological methods and proteomics and genomic arrays, are generally understood by those skilled in the art.

  The array provides for detection and identification of pathogens and virulence factors as well as detection and identification of transformed cells and tissues using samples from subjects. Information about a patient's disease state, i.e., a patient data set, is obtained by first obtaining a blood sample from a subject and then isolating DC from the blood sample to obtain one or more of the described APC arrays. Obtained using. For example, the described hybridization array is used to compare a patient's DC signature to one or more control DC signatures. Control signatures on the array minimally show both normal or healthy DC signatures and abnormal or pathological DC signatures for one or more disease states. Various other embodiments include additional control DC signatures that provide reference signatures for stages of various disease states, eg, stages of cancer. The resulting arrays and datasets are used, for example, to discover or diagnose the presence of genetic diseases or chromosomal abnormalities, or to provide information about identity, inheritance or suitability to diagnose a predisposition to a disease or condition, To determine the contact and identification of biological or chemical weapon samples or toxic industrial chemicals to diagnose or diagnose neoplastic transformation of cells or tissues to diagnose infection by pathogenic microorganisms Useful.

  In one aspect, APC arrays designed to identify the presence or absence of specific pathogens and the ongoing immunological consequences of the disease states they are associated with have been developed. For example, an array is created that detects and monitors viral infections such as HIV. Arrays are consensus APC signatures from immature or naive APC, APC from HIV contact but asymptomatic, APC from HIV contact and early symptomatic person, and APC from late symptomatic person including. Similar virus arrays are developed, such as those useful for diagnosing and monitoring hepatitis, neoplastic viruses or other chronic or pathogenic viral infections. Diagnostic arrays that can be used to monitor viral vectors used for gene therapy, such as for vaccinia or poxviruses, and more particularly should produce a DC signature that is different from the wild type vector, Those specific for the transformation vector are also preferred. In yet another aspect, the array comprises DC and macrophage cells contacted with human APCs, particularly CDC priority list pathogens. Such types of arrays facilitate rapid emergency diagnosis, etiology research, response and treatment of individuals in contact or potential contacts. Since a DC array specific for biological weapon pathogens provides rapid detection and response of terrorist or enemy biological weapon attacks, an array specific for mainland defense or military use is also provided herein. Such arrays include smallpox arrays, Bacillus anthracis arrays and other WMD pathogens. For example, human APCs, particularly DC and macrophage cells, contacted with toxic pathogens facilitate emergency diagnosis, response and treatment of individuals who have contacted or may have been contacted. In another embodiment, the arrays and patient data sets obtained therefrom facilitate forensic or toxicological studies of the contacted individual.

  In another aspect, the array is obtained from human APCs, particularly DCs and macrophages from patients with different tumors, including different stages of tumor growth. The APC array is designed to identify the presence or absence of specific tumor antigen markers and the immunological outcome of the patient's tumor during the progression of the patient's cancer. This type of array facilitates rapid diagnosis, tumor identification and appropriate treatment of affected individuals. Each of the following cancer types produces a specific APC response and may be detected using the techniques described: acute lymphoblastic leukemia, adults; acute lymphoblastic leukemia, children; acute myeloid Leukemia, adult; acute myeloid leukemia, child; adrenocortical cancer; adrenocortical cancer, child; AIDS-related cancer; AIDS-related lymphoma; anal cancer; astrocytoma, childhood cerebellum; astrocytoma, childhood cerebrum; Bladder cancer; bladder cancer, child; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma, child; brain tumor, adult; brain tumor, brain stem glioma, child; brain tumor, cerebellar astrocytoma, child Brain tumor, cerebral astrocytoma / malignant glioma, child; brain tumor, ependymoma, child; brain tumor, medulloblastoma, child; brain tumor, supratentorial primitive neuroectodermal tumor, child; brain tumor, visual pathway and hypothalamic glioma , Children; brain tumor, children (others); breast cancer; breast Breast cancer, children; breast cancer, males; bronchial adenoma / carcinoid, children; carcinoid tumors, children; carcinoid tumors, gastrointestinal tract; cancer, adrenal cortex; cancer, islet cells; cancer of unknown primary site; Primary; Cerebellar astrocytoma, children; cerebral astrocytoma / malignant glioma, children; cervical cancer; childhood cancer; chronic lymphocytic leukemia; chronic myelogenous leukemia cells; chronic myeloproliferative disease; clear cell sarcoma of tendon sheath Colon cancer; colorectal cancer, children; cutaneous T-cell lymphoma; endometrial cancer; ependymoma, children; epithelial cancer, ovary; esophageal cancer; esophageal cancer, children; Ewing tumor; Extragonadal germ cell tumor; extrahepatic cholangiocarcinoma; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; gastric (stomach) cancer; stomach (stomach) cancer, children; Gastrointestinal carcinoid tumor; germ cell tumor, extracranial, child; embryo Alveolar tumor, extragonadal; germ cell tumor, ovary; gestational choriocarcinoma; glioma, childhood brain stem; glioma, childhood visual pathway and hypothalamus; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer, adult (primary) ); Hepatocellular (liver) cancer, childhood (primary); Hodgkin lymphoma, adult; Hodgkin lymphoma, childhood; Hodgkin lymphoma, during pregnancy; hypopharyngeal cancer; hypothalamic and visual conduction glioma, childhood; intraocular melanoma Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; kidney cancer; laryngeal cancer; laryngeal cancer, children; leukemia, acute lymphoblastic, adult; leukemia, acute lymphoblastic, child; leukemia, acute myeloid, adult Leukemia, acute myeloid, children; leukemia, chronic lymphoid; leukemia, chronic myeloid; leukemia, hairy cells; lip and oral cancer; liver cancer, adult (primary); liver cancer, child (primary); lung cancer , Non-small cell, lung cancer, Small cells; lymphoblastic leukemia, adult acute; lymphoblastic leukemia, childhood acute; lymphocytic leukemia, chronic; lymphoma, AIDS related; lymphoma, central nervous system (primary); lymphoma, cutaneous T cell; lymphoma, Hodgkin, adult; lymphoma, Hodgkin, child; lymphoma, Hodgkin pregnant; lymphoma, non-Hodgkin, adult; lymphoma, non-Hodgkin, child; non-Hodgkin pregnant; lymphoma, primary central nervous system; macroglobulinemia, Waldens Male breast cancer; malignant mesothelioma, adult; malignant mesothelioma, child; medulloblastoma, child; melanoma; melanoma, intraocular; Merkel cell tumor; mesothelioma, malignant; metastasis of unknown primary Squamous cell carcinoma of the cervix; multiple endocrine neoplasia syndrome, children; multiple myeloma / plasmacytoma; mycosis fungoides; myelodysplastic syndrome; myelodysplastic / myeloproliferative disorder; myeloid leukemia, chronic; White Disease, adult acute; myeloid leukemia, childhood acute; melanoma, multiple; myeloproliferative syndrome, chronic; nasal and sinus cancer; nasopharyngeal cancer; nasopharyngeal cancer, child; neuroblastoma; non-Hodgkin lymphoma, adult Non-Hodgkin lymphoma, children; non-Hodgkin lymphoma, during pregnancy; non-small cell lung cancer; oral cancer, children; oral and lip cancer; oropharyngeal cancer; osteosarcoma / bone malignant fibrous histiocytoma; ovarian cancer, children; Ovarian epithelial cancer; ovarian germ cell tumor; low-grade ovarian cancer; pancreatic cancer; pancreatic cancer, children; pancreatic cancer, islet cell; paranasal sinus and nasal cavity cancer; parathyroid cancer; And supratentorial primitive neuroectodermal tumor, children; pituitary tumor; plasmacytoma / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non-Hodgkin lymphoma; primary central nervous system lymphoma Primary liver cancer, adult Human; primary liver cancer, children; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal cell carcinoma, children; renal pelvis and ureter, transitional cell carcinoma; retinoblastoma; rhabdomyosarcoma, children; salivary gland Cancer; salivary gland cancer, children; sarcoma, Ewing tumor; sarcoma, Kaposi; sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; sarcoma, rhabdomyosarcoma, children; sarcoma, soft tissue, adult Tissue, child; Sezary syndrome; skin cancer; skin cancer, child; skin cancer (melanoma); skin cancer, Merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma, adult; soft tissue sarcoma, child; Squamous cell carcinoma of the cervix, metastatic; stomach (stomach) cancer; stomach (stomach) cancer, children; supratentorial primitive neuroectodermal tumor, children; T cell lymphoma, skin; testicular cancer; thymoma, children; thymoma and Thymic cancer Thyroid cancer; Thyroid cancer, Children; Localized transitional cell carcinoma of the renal pelvis and ureter; Chorionic tumor, during pregnancy Primary site unknown, adult cancer; primary site unknown, childhood cancer; childhood rare cancer; ureter and renal pelvis, transitional cell cancer; ureteral cancer; uterine cancer, endometrium; uterine sarcoma; Tract and hypothalamic glioma, children; vulvar cancer; Waldenstrom macroglobulinemia; and Wilms tumor.

  An array that provides detection and monitoring of various cancers such as breast cancer, colon cancer, ovarian cancer, uterine cancer, prostate cancer, glioma, melanoma, small and large cell cancer, leukemia and other neoplastic and precancerous disease states Is produced. Abnormally glycosylated MUC-1 or markers such as CEA or hepsin are examples of common tumor markers known to be associated with most of the tumors described above. Comprehensive lists containing tumor specific markers are known in the medical literature. Exemplary arrays include APC-derived consensus APC signatures obtained from patients with immature or naïve APC-derived and stage 0, 1, 2, 3 or 4 staged tumors. Histological profiles and other medical data can be used in conjunction with the APC array to provide additional information about the disease state. The flexibility and specificity of the APC response to cancer makes it possible to identify cancer types with high specificity and determine the stage of the disease. Thus, they also allow health professionals to monitor the course of therapy, for example by monitoring changes in APC signatures during chemotherapy. For example, gemcitabine is provided to a pancreatic cancer patient, and DC is extracted from the patient before or during a gemcitabine treatment course and used with a pancreatic cancer DC array. The array indicates that pancreatic cancer is stage 3 at the start of treatment, indicating that 1 month of gemcitabine treatment has returned the cancer to stage 2 stage, thereby indicating continued continuation of the patient's gemcitabine treatment.

  In yet another embodiment, APC arrays are made from individuals with a genetic disorder. Typical genetic diseases include, for example, the presence of a gene, a gene expression product that is a bioactive molecule that causes or contributes to a disease state, or a gene expression product in a healthy individual improves or prevents the disease state Disease states caused by a deficiency of a gene that is a physiologically active molecule. The former example is cystic fibrosis, and the disease state is caused by mutations in the CFTR protein. An example of the latter is PKU, where the disease state is caused by a deficiency in an enzyme that allows the metabolism of phenylalanine. Examples of genetic diseases suitable for screening using the assays and methods of the present invention include, for example, multiple sclerosis, endocrine diseases, Alzheimer's disease, amyotrophic lateral sclerosis, lupus, Angelman syndrome, Charcot-Marie Tooth disease, epilepsy, essential tremor, fragile X syndrome, Friedreich ataxia, Huntington's disease, Niemann-Pick disease, Parkinson's disease, Prader-Willi syndrome, Rett syndrome, spinocerebellar atrophy, William syndrome, Ellis-fan- Krefeld syndrome, Marfan syndrome, myotonic dystrophy, cerebral white matter atrophy, atherosclerosis, Vest disease, Gaucher disease, glucose galactose malabsorption, brain atrophy, juvenile diabetes, obesity, paroxysmal nocturnal hemoglobin Includes urine disease, phenylketonuria, refsum disease and tanger disease That. Such arrays are useful in detecting a patient's genetic disease and monitoring the genetic disease patient during treatment. Similarly, the assays of the present invention monitor the course of gene therapy by monitoring the immune status of patients being treated in this way, particularly for the appearance of healthy gene products or for adverse reactions to gene therapy vectors. .

  While the above discussion focuses on the use of APC in diagnostics to measure biological changes in a sample, in another embodiment, blood, sputum, urine, saliva, mucus, cerebrospinal fluid, A direct analysis of a subject's body fluid, such as lymph fluid, can be subjected to the assay. These samples are analyzed using a variety of medical techniques, e.g., experimental techniques such as lumbar puncture or proteomics tools to look for infections in cerebrospinal fluid, e.g. mass spectrometry, and are generally known below. Is also described. Thus, the assay of the present invention may involve monitoring APC for changes in conjunction with direct analysis of a subject's biological fluids that provide a more sophisticated detection and monitoring method.

  Thus, APC provides a highly specific and rapid means of monitoring biological changes in an organism based on specific genomic and proteomic signatures typified by DCs in a particular state. The above considerations are centered on the use of APC in assays that use such general techniques as hybridization or immunological reactivity. Other proteomic tools are suitable for measuring changes in APC status. A particularly preferred method of obtaining DC proteomic signatures involves obtaining a mass spectrum of an APC sample.

  In the last decade, mass spectrometry (MS) has become an important analytical tool in the analysis of biopolymers. Mass spectrometry provides a means of “weighing” individual molecules by ionizing the molecules under vacuum and “flying” them by evaporation. Under the influence of a combination of electric and magnetic fields, the ions orbit according to their individual mass (m) and charge (z). To perform MS, the sample under study is subjected to energy desorption / ionization (EDI) from the surface by energy injection. Typically, EDI is a thermal desorption / ionization (TDI), plasma desorption / ionization (PDI) and various types of irradiation desorption / ionization (IDI) such as fast atom bombardment (FAB), electron bombardment, etc. is there. If a laser is used to ionize the sample, the method is called laser desorption / ionization (LDI), such as matrix-assisted laser desorption / ionization (MALDI). Desorption can preferably be assisted by presenting the MS analyte with various helper substances or functional groups on an ionized surface such as surface enhanced laser desorption / ionization (SELDI).

  For low molecular weight molecules, mass spectrometry has long been part of the normal physico-organic repertoire for analyzing and characterizing organic molecules by determining the mass of the parent molecular ion. With the introduction of so-called “soft ionization” methods, ie MALDI and electrospray ionization (ESI), intact ionization, detection and accurate masses of macromolecules, such as peptides and proteins with masses above 300 kDa Measurement has become possible (see Fenn, JB, et al., (1989) Science 246, 64-71; Karas M. & Hillenkamp F. (1988) Anal. Chem. 60, 2299-3001). In addition, by arranging collisions between the ionized parent molecule and other particles (for example, argon atoms), the ionized parent molecule is fragmented and secondary ions are formed by collision-induced dissociation (CID). Fragmentation patterns / paths can very often lead to more detailed information, for example molecular structural information.

  MALDI-MS and ESI-MS have been used to analyze nucleic acids and proteins (see Nordhoff E., et al., (1997) Mass Spectrom. Rev. 15: 67-138). However, since nucleic acids are highly polar biomolecules and difficult to volatilize, there is an upper limit on mass for clear and accurate resolution. ESI appears to be superior to MALDI for intact detachment of large nucleic acids in the MDa mass range (Fuerstenau SD & Benner WH (1995). Rapid Commun. Mass Spectrom. 9, 1528-38; Chen R., Cheng X., Mitchell et al., (1995). Anal. Chem. 67, 1159-1163).

  Several reports of MALDI-MS of large DNA molecules using ultraviolet (UV) lasers have been reported (Ross PL & P. Belgrader (1997) Anal. Chem. 69: 3966-3972; Tang K., et al ., (1994) Rapid Commun. Mass Spectrum. 8: 727-730; Bai J., et al., (1995) Rapid Commun. Mass Spectrum. 9: 1172-1176; Liu YH-, et al., (1995 ) Anal. Chem. 67: 3482-3490 and Siegert CW, et al., (1997) Anal. Biochem. 243, 55-65, but based on these reports, the mass of nucleic acids exceeding 30 kDa Analysis by UV-MALDI-MS is clearly more difficult at the current upper mass limit of about 90 kDa (Ross PL & P. Belgrader (1997) Anal. Chem. 69: 3966-3972). The degradation in the quality of the UV-MALDI spectrum was due to a combination of ion fragmentation of the phosphate backbone and numerous salt formations, since RNA is much more stable than DNA under UV-MALDI conditions. The available mass range is up to about 150 kDa (Kirpekar F., et al., (1994). Nucleic Acids Res. 22, 3866-3870).

  Analysis of nucleic acids by IR-MALDI using a solid matrix (mostly succinic acid, to a lesser extent urea and nicotinic acid) has been described (Nordhoff, E. et al., (1992) Rapid Commun. Mass Spectrom. 6: 771-776; Nordhoff, E. et al., (1993) Nucleic Acids Res. 21: 3347-3357; and Nordhoff, E. et al., (1995) J. Mass Spec. 30: 99-112) . A 1992 paper by Nordhoff et al. Reported that 20-mer DNA and 80-mer RNA are almost the upper limit of resolution. However, the 1993 Nordhoff et al paper provides separate spectra of 26mer DNA and 104mer tRNA. The 1995 Nordhoff et al paper shows that UV-MALDI using a 3-hydroxypicolinic acid solid matrix gives a substantially better spectrum of 40mer analysis than IR-MALDI using succinic acid (Figure 1). (See (d) and 1 (e)). In fact, a 1995 report reports that IR-MALDI provided a substantial degree of rapid fragmentation.

  In a time-of-flight (TOF) mass spectrometer, the ion mass to charge ratio m / z can be determined from the time of flight. In mass spectrometry, what is always measured is the mass-to-charge ratio m / z, where m is the mass, and z is the number of basic charges carried by the ion. , Mentions only the mass m and its measurement. Since many types of ionization, such as MALDI, mainly supply only a single charged ion (z = 1), the difference between these types of ionization actually disappears. A time-of-flight mass spectrometer (TOF-MOS) with an ion selector and velocity focusing reflector can measure the daughter ion or fragment ion spectrum of the parent ion selected by the ion selector based on the time of flight. is there. Degradation of parent ions to daughter or fragment ions can be done by introducing excess energy during ionization (so-called PSD “post source decay” spectrum) or other methods such as collision-induced fragmentation Can be induced. The parent and daughter ions generated by the decomposition enter the reflector at the same average speed but different mass proportions at the same time so that they are dispersed according to mass in the reflector by different energies.

  Thus, mass spectrometry and other tools that allow detection of, for example, infrared and ultraviolet absorption spectra, nuclear magnetic resonance spectra, and biomolecular interaction analysis (e.g., ELISA or surface plasmon resonance (SPR) profiles, Nedlekov et al , (2003) Appl. Env. Microbiol.) And other techniques for measuring physical properties of samples also provide methods for analyzing samples. The information obtained from the method using APC described above in relation to conventional experimental methods provides an integrated approach that can resolve the different properties of each sample under consideration. For example, if the MS profiles of two samples show a highly similar pattern, a second analysis such as IR spectrum, NMR spectrum or SPR is used to provide additional comparison signatures and information. The results provide independent identification of the sample (single or mixed state), and in some embodiments also provide qualitative information such as quantitative information of the sample such as concentration and identification of other drugs or substances in the sample mixture An analysis signature profile specific to each sample that can be or the sample under analysis.

  A preferred method uses mass spectrometry to obtain a proteomic signature of APC in response to health status and antigen addition. Mass spectrometry can also be used directly on mixtures suspected of containing antigens or other contaminants. The most preferred analytical methods for obtaining these signatures include SELDI such as ProteinChip® System Series 4000 from Ciphergen or MALDI such as prOTOF ™ 2000 MALDI O-TOF mass spectrometer from Perkin Elmer. MALDI O-TOF based on an orthogonal platform that connects MS to MS.

  In one aspect, the proteomic signature of the APC, preferably DC, is obtained after loading with toxins and organisms from the National Institute for Allergy and Infectious Diseases Biodefense Priority Pathogens List . DCs are cultured with the antigen or fragment thereof as described below. Get a proteomic signature. These related antigens include Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Variola major (pox) and other poxes. Viruses, Francisella tularensis (wild boar), and viral hemorrhagic fever, LCM, Junin virus, Machupo virus, Guanarito virus and Lassa fever Arenaviruses such as those that arise, Bunyavirus and Hantavirus such as those that produce Rift Valley fever, Calicivirus, Hepatitis A, B and C, West Nile virus ( West Nile Virus, LaCrosse, California encephalitis, VEE, EEE, WEE, Japanese Encephal virus viral encephalitis such as itis virus, Kyasanur Forest Virus, Tickborne hemorrhagic fever virus, Crimean-Congo Hemorrhagic fever virus, tick-borne virus Encephalitis virus (Tickborne encephalitis viruses), yellow fever, multidrug resistant TB, influenza, other rickettsia and rabies, Flavirus, Dengue, Filovirus, Ebola, Marburg Burkholderia pseudomallei, Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (nasal fin), ricin toxin (Ricinus communis), Clostridium perfringens ε toxin, Staphylococcus enterotoxin B Typhus fever (Rickettsia prowazekii), food and waterborne pathogens, Diarrheagenic E.coli, pathogenic Vibrios, Shigella species, Salmonella ), Bacteria such as Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, and Cryptosporidium parvum, Cyclospora kaytanensis caya, Included are protozoa such as Giardia lamblia, Entamoeba histolytica, Toxoplasma, and Microsporidia. These biological samples can be detected and identified based on criteria such as lipopolysaccharide content, glycoprotein content, membrane composition, presence of viral envelope, expression of specific proteins such as virulence factors and other biochemical profiles To. See, for example, Dell A, Morris HR, Glycoprotein structure determination by mass spectrometry. Science. 2001; 291 (5512): 2351-6. Rudd PM et al., Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc Natl Acad Sci US A. 1999, 96 (23): 13044-9 reference. In addition, Beerman et al., The lipid component of lipoproteins from B. burdorferi: structural analysis, antigenicity, and presentation. by human dendritic cells), Biochem. Biophys. Res. Comm., 267: 897-905 (2000). Analytical signatures are subjects who have (or are suspected to contact) one or more toxins and organisms from the National Institute for Allergy and Infectious Diseases Biodefense Priority Pathogens List. Cell, body fluid and tissue samples. The DC and fluid or tissue signatures are compared to the reference signature to confirm contact and aid in monitoring treatment. For example, a blood sample is obtained from a subject suspected of contacting smallpox. Divide the sample in two; collect DC from one and purify plasma from the other. Both samples are subjected to mass spectrometry. Compare the reference signature with a DC signature that provides positive and negative controls for contact and naive DC and assay the plasma for the presence of variola virus. The signature can confirm the infection and promote quarantine before the patient becomes viremia or symptomatic.

  SELDI or MALDI O-TOF mass spectrometer signatures can profile nucleic acid, proteins, carbohydrates and lipids in microbial samples, but preferably can profile and obtain the pathogen-wide signature. Signatures are microbial species and species variants such as E. coli O157, microbial growth phase, e.g. sporulation, vegetative or active growth phase and relative age and pathogenicity, e.g. pyogenic exotoxin A production of group A streptococci, Other features are identified, such as enterotoxin production in Vibrio choleae cholera toxin, Shiga toxin-producing E. coli (STEC) or enterohemorrhagic strain (EHEC) in E. coli. For example, Lai et al. Identified 67 strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum. We consider that we investigated by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a 6 month period were also examined. The results were analyzed by multivariate analysis and compared with the results of previous quantitative expression studies, pyrolysis mass spectrometry (PyMS) studies and studies using DNA-DNA and 16S rRNA sequence homology. A taxonomic relationship between FT-IR data and these studies was observed. It was observed that culture age had little effect on the resulting spectrum. A reproducibility study showed that there was an association between spectra generated twice in a 6 month period. Lai et al. Conclude that FTIR is a reliable method for studying Carnobacterial classification and has a further potential as a rapid method for use in Carnobacterium identification. It was. Lai, S., R. Goodacre, et al. (2004). “Whole-organism fingerprinting of Carnobacterium using Fourier transform infrared spectroscopy (FT-IR). the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR).) ”Syst Appl Microbiol 27 (2): 186-91. Similarly, Lee et al. Discuss bacterial analysis methods combined with flow field flow fractionation (flow FFF) separation techniques using detection by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The composition of the carrier liquid used for the flow FFF is selected based on retention of bacterial cells and compatibility with MALDI methods. A combination of Flow FFF and MALDI-TOF MS has been demonstrated for P. putida and E. coli. Whole cell fractions were collected by separation with FFF and further analyzed by MALDI-MS. Each fraction collected at different time intervals corresponded to different sizes and different growth phases of bacteria. Lee, H., SK Williams, et al. (2003). "Analysis of whole bacterial cells by flow field". Flow field flow fractionation and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. -flow fractionation and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.) "Anal Chem 75 (11): 2746-52. Similarly, Lefmann et al. Discuss MALDI-TOF MS after base-specific cleavage of a 16S rRNA gene (rDNA) that has been PCR-amplified and transcribed in vitro, used to identify actinomycetes. The full length 16S rDNA reference sequence of 12 standard strains of Mycobacterium spp. Was frequently isolated from clinical specimens was determined by PCR, cloning and sequencing. For comparative sequence analysis based on MALDI-TOF MS, we used 12 standard strains and 24 clinical isolates of actinomycetes 16S rDNA signature sequences (approximately 500 bp) using RNA promoter-tagged forward primers. PCR amplification. T7 RNA polymerase-mediated transcription of the forward strand in the presence of 5-methylribo-CTP maximized the mass difference of the fragments produced by base-specific cleavage. The in vitro transcript was then treated with RNase T1 to generate G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 standard strains, allowing unquestionable identification. All 24 clinical isolates were unambiguously identified by comparing the detected mass signal pattern with a computer pattern derived from the reference sequence of the standard strain and the reported 16S rDNA sequence. The 16S rDNA microheterogeneity of the Mycobacterium xenopi standard strain (DSM 43995) was detected by MALDI-TOF MS and subsequently confirmed by Sanger dideoxy sequencing. Analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts was rapid for Mycobacterium tuberculosis group and ubiquitous actinomycetes (actinomycetes other than tuberculosis), Lefmann et al. Conclude that it enables reliable identification. Lefmann, M. et al., Novel mass spectrometry-based tool for genotypic identification of mycobacteria. J Clin Microbiol 42 (1): 339-46 (2004) reference. Thus, one object of the present invention includes obtaining proteomics, genomes, lipids, carbohydrates and whole organism signatures of bacterial pathogens. Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry alone or in combination with other assays. Microbial identification is not limited to bacteria, and thus other pathogenic organism analysis signatures include those of fungi, viruses, prions and other infectious fungi and pathogens.

  Proteomic signatures derived from APC and those obtained by direct assessment of pathogens in patient fluids are used in the diagnosis of the described diseases, e.g., terbinafine, fluconizole, lamivudine, ciprofloxacin, vancomycin, It is particularly useful for monitoring the course of treatment in response to antimicrobial compounds such as penicillin, methicillin and other antibiotics. The tissue, fluid and cell signatures of subjects treated therapeutically with antimicrobial compounds can also be analyzed for toxicity during such treatment. Detection of viral samples is described, for example, in Hong et al., Who assayed for hepatitis B virus (HBV) mutations that allow lamivudine resistance to occur during long-term treatment with lamivudine. Treatment with lamivudine often results in the selection of HBV virions that have amino acid substitutions in the YMDD motif of HBV DNA polymerase. MALDI-TOF MS genotyping detects HBV variants sensitively and specifically. The Hong et al. Assay is based on PCR amplification and mass measurement of oligonucleotides containing mutation sites in the YMDD motif. The genotyping based on MALDI-TOF MS described there is sensitive enough to detect as few as 100 copies of HBV genome per milliliter of serum and measures a mixture of wild type and variant viruses It has excellent specificity. When analyzing sera from 40 patients, an assay based on MALDI-TOF MS accurately identified known virus variants and additional virus pseudo-species not detected by previous methods, and their relative abundance . Sensitivity, accuracy, and high-throughput analysis possibilities make assays based on MALDI-TOF MS suitable for mass screening of HBV-infected patients receiving lamivudine to better understand disease progression and response to treatment Hong et al. Conclude that it can help. See Hong et al., Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments, J Hepatol. (2004). Accordingly, one object of the present invention includes proteomics of viral pathogens, genomes, lipids, carbohydrates and whole organism signatures as well as analysis signatures of DCs and other tissues, body fluids and cells of subjects with viral infection. Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry alone or in combination with other assays.

  Bonetto et al. Describe the structure and biological nature of prion protein scrapie (PrP (Sc)) as a mechanism for the structural transition of cells (PrP (C)) to disease-specific isoforms and as a basis for pathogenesis of prion diseases. The evaluation of characteristics is considered. Bonetto et al. Observed that a 106 amino acid construct derived from mouse PrP (called PrP106 or miniprion) is highly toxic to primary neuronal cultures and induces a significant increase in membrane microviscosity. See Bonetto V. et al., Synthetic miniprion PrP106, J Biol Chem. 277 (35): 31327-34 (2002). Thus, in yet another aspect, the present invention includes prion sample signatures and subject APC and tissue, body fluid and cell signatures in which prion infection is observed. Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry alone or in combination with other assays.

  Yet another object of the present invention is to provide healthy subjects and various stages of physical degeneration such as myocardium, kidney or neural tissue, infection of various stages such as viruses or bacteria, or various stage traits. Obtaining signatures such as SELDI or MALDI O-TOF MS and other analytical signatures from diseased subjects characterized by conversion, malignancy or tumorogenicity, ie APCs, body fluids and tissues. In particular, cancer and pre-malignant tissues all undergo large biochemical changes compared to unaffected cells and tissues and can be easily detected by spectral and other types of analytical methods. An example of this is the altered glycosylation pattern seen in the tumor-associated antigen MUC-1 of a number of different cancer types or chorioembrionic antigen (CEA) or retinoblastoma (RB), p53 and cyclin-dependent kinases It is the differential expression of tumor suppressor genes such as cdk. Numerous markers of cell transformation and cancer are known in the medical literature, all of which can be disease signatures for the purposes of the present invention. Similarly, these tissues exhibit changes in metabolic status in response to treatment with chemotherapy samples and radiation. These changes are molecular signatures of response to therapy and are therefore useful for the purposes described herein. Thus, the methods of the invention can be used, for example, to identify specific tumor types, determine the stage, and monitor changes in a tumor over the course of treatment, such as chemotherapy or radiation. The methods described can also be used to monitor changes in healthy organs and tissues that occur during such chemotherapy or radiation therapy, for example, to assess systemic toxicity of the treatment to regulate the course of treatment. it can. In one aspect, a toxicity profile for chemotherapy is provided. This profile includes tissue specific molecular analysis signatures of multiple mammalian organs and tissues in an untreated state, ie not in contact with a chemotherapeutic agent and in response to multiple doses of the drug. The profile may include a dose response signature of the tissue over the time domain, i.e. over a period of time. Thus, the present invention includes analytical signatures useful for disease detection and treatment. Chaurand et al. Determined that analysis of organ thin tissue sections yielded over 500 individual protein signals in the mass range of 2 to 70 kDa that directly correlated to protein composition within specific regions of tissue samples. Such profiling involving imaging MS has been applied to a number of affected tissues including human glioma and non-small cell lung cancer. Investigation of the resulting complex MS dataset has identified disease state and patient prognosis specific protein patterns. See Chaurand P et al., Assessing protein patterns in disease using imaging mass spectrometry. J Proteome Res. 2004 Mar-Apr; 3 (2): 245-52. Similarly, Ahmed et al. Discuss proteins that are differentially expressed in the serum of ovarian cancer patients that may be useful as biomarkers for ovarian cancer. In Ahmed's paper, a total of 24 serum proteins were differentially expressed in Grade 1 ovarian cancer patients, 31 were differentially expressed in Grade 2, and 25 were differentially expressed in Grade 3. Six of the protein spots that were significantly up-regulated in all groups of ovarian cancer patients were nanoelectrospray quadrupoles as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum It was identified by time-of-flight mass spectrometry (n-ESIQ (q) TOFMS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Further identification of different pathological grade spots was confirmed by Western blotting and immunohistochemical localization using monoclonal antibodies against the haptoglobin epitope contained in HAP1. See Ahmed N et al., Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer. Br. J. Cancer 2004. Similarly, Bharti et al. Consider the detection of serum tumor biomarkers at an early stage to improve the overall survival of cancer patients. Using protein identification techniques based on MALDI-TOF mass spectrometry (MS), SCLC-specific overexpressed proteins are identified as haptoglobin α-subunits, whose serum levels correlate with disease stage. ing. The average level of α-haptoglobin was increased in SCLC serum compared to normal controls. Serum HGF was also investigated as a potential tumor biomarker and found to be associated with disease state. Anticancer Res. 2004 Mar-Apr; 24 (2C): 1031-8. Some other tumor types are subject to detection using the methods of the invention. Iwadate et al. Consider glioma detection and response to chemotherapy treatment. The biological characteristics of glioma, characterized by heterogeneous biological aggressiveness in the same histological category, are accurately described by global gene expression at the protein level. Iwadate et al. Investigated that proteomic analysis based on two-dimensional gel electrophoresis and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry can identify differences in protein expression between high-grade and low-grade glioma tissues . Proteomic profiling patterns were compared in 85 tissue samples: 52 glioblastoma multiforme, 13 anaplastic astrocytomas, 10 astrocytomas and 10 normal brain tissues. Iwadate et al. Were able to fully distinguish normal brain tissue from glioma tissue by cluster analysis based on proteome profiling patterns. Proteome-based clustering was significantly correlated with patient survival, and Iwadate et al. Were able to identify a biologically distinct subset of astromas with aggressiveness. Iwadate et al. Found that discriminant analysis extracted a set of 37 proteins that were differentially expressed based on histological grading. Of those, many of the proteins that were increased in high-grade gliomas were classified as signaling proteins, including small G proteins. Immunohistological analysis confirmed the expression of the identified proteins in glioma tissue. Iwadate Y, et al. Molecular classification and survival prediction in human gliomas based on proteome analysis. Cancer Res. 2004 Apr 1; 64 (7): 2496- See 501. Friedman and colleagues consider mass spectrometry (MS) combined with two-dimensional difference gel electrophoresis analysis used to investigate tumor-specific changes in human colorectal cancer and adjacent normal mucosal proteome is doing. Friedman et al. Examined more than 1500 protein spot features using DIGE technology with a sample internal standard mixture in each of a pair of normal / tumor comparisons, and statistics of each protein amount change between multiple samples. Significant quantitative comparisons were made simultaneously. Matrix-assisted laser desorption / ionization time-of-flight and tandem (TOF / TOF) MS provide sensitive, accurate mass spectral data for database interrogation, with quantities varying between cohorts We identified 52 unique proteins (including proteolytic and posttranslationally modified isoform redundancy). Friedman DB et al., Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics. 2004 Mar; 4 (3): See 793-811. Hamler et al. Consider a two-dimensional liquid phase separation scheme combined with mass spectrometry (MS) for proteomic analysis of cell lysates of normal and malignant breast cancer epithelial cell lines. Liquid phase separation consists of isoelectric focusing as the first dimension and non-porous silica reverse phase high performance liquid chromatography (NPS-RP-HPLC) as the second dimension. Protein quantification and mass measurements are performed using electrospray ionization time-of-flight MS (ESI-TOF MS). Proteins are identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight MS and MALDI quadrupole time-of-flight (QTOF) tandem mass spectrometry (MS / MS). Hamler et al. Created a mass map that allows visualization of protein differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins obtained from mass mapping experiments in the limited pH range, 40 (36%) were identified as positive by peptide mass fingerprinting and assigned to mass map bands. Of these 40 species, 22 were more strongly expressed in one or more of the malignant cell lines. These proteins represent breast cancer biomarkers that may aid in diagnosis, treatment or drug development. Hamler RL, et al., A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification ). Proteomics. 2004 Mar; 4 (3): 562-77. Veestra et al. Consider serum protein fingerprinting. Numerous proteomic studies have focused on the identification and subsequent comparative analysis of thousands of proteins that occupy complex biological systems such as serum and tissues. See Veenstra T.D et al., Serum protein fingerprinting. Curr Opin MoI Ther. 2003 Dec; 5 (6): 584-93. Thus, the spectroscopic profiles or patterns of these proteomics, carbohydrates, nucleic acids and lipids provide numerous tissue signatures in both healthy and disease states, and from a diagnostic point of view the presence of disease, Can be used to monitor changes in organisms having In one embodiment, serum or lymph samples are used to obtain such signatures. In another embodiment, DC is used. In other embodiments, the APC provides an analytical signature. In yet other embodiments, blood cells, muscle tissue, nerve tissue, epithelial tissue and connective tissue are evaluated.

  Another object of the present invention is as a result of chemical signatures of toxic industrial chemicals as well as APCs, tissues, body fluids and cells of a subject in contact with or suspected of having contacted toxic industrial chemicals (TIC). Includes determination of resulting proteomics, genome, lipid and carbohydrate signatures. Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry alone or in combination with other analytical methods. The resulting signature is stored in a database and made available for diagnostic and therapeutic applications. Toxic industrial chemicals are generally understood as materials with toxicity (LC50 by inhalation) of less than 100,000 Mg / min / M3 and appreciable (undefined) vapor pressure at 20 ° C. As used herein, the term TIC generally includes toxic industrial chemical materials (TIM) that are considered to be any substance that produces a toxic effect in an individual in contact with a given amount of inhalation, ingestion or absorption. Examples of TIC and TIM include fuels, oils, pesticides and herbicides, acids and bases, radiation sources, fertilizers, arsenic, chlorine, bromine, carbon disulfide, cyanide, metals (e.g., cobalt, lead, mercury, Cadmium and thallium), phosgene and other organic and heavy metal toxins. Numerous TICs and TIMs are known in the industry, and the materials mentioned above are not intended to be comprehensive or limiting.

  In another aspect, the present invention relates to signatures of substances important to defense such as biological and chemical weapons (also known as WMD), such as chemical, proteomics, genomes, lipids, carbohydrates and whole organism signatures, and such Provide APC, tissue, body fluid and cell proteomics, genome, lipid and carbohydrate signatures of subjects in contact with or suspected of contacting the substance. Preferably, these signatures are obtained using SELDI, MALDI O-TOF MS and other analytical methods. In particular, since spectral analysis provides a fast and accurate means of detection, it is possible to use the present invention as part of a rapid or initial reaction program for field identification of biological and chemical weapons in a sample.

  The first stage of the analytical method involves obtaining a sample of the factor (TIC, WMD) bacteria, virus, prion, cell, APC, fluid or tissue under study. Samples can be processed prior to investigation, i.e., dissolved in water or solvent, or used intact. Sample analysis methods can be used to obtain rudimentary information about the sample. The collection of mass spectra and their analysis are as follows. Apply the sample to the inlet port of the MS, and the mixture or whole cell (or organism) may contain lipid, carbohydrate, nucleic acid and / or peptide structures or any other inorganic or organic structure If it contains more than one analyte. The sample may be processed prior to MS, the sample may be converted to one in which the MS analyte is a derivative of the starting analyte, and the amount of non-analyte species is altered compared to the starting sample. The relative presence of different MS analytes in the sample is altered compared to the starting sample, and the concentration of the MS analyte is altered compared to the corresponding starting analyte in the starting sample; Alternatively, sample components such as solvents have been changed and / or the analyte has been changed from a dissolved form to a solid form, eg, a co-crystallized form. Such processing includes, for example, digestion into fragments of various sizes and / or chemical derivatization of the analyte. Digestion may be purely chemical or enzymatic. Derivatization includes the so-called mass tag method of starting analytes or fragments or other derivatives formed during sample processing protocols. Other processing involves purifying and / or concentrating the sample prior to analysis. Such treatment applies for example to analytes that are biopolymers containing carbohydrate, lipid, nucleic acid and / or peptide structures. Alternatively, the sample may pass through the microchannel structure without modification.

Example 1
The following examples describe in detail the use of APC as a disease biosensor. Reference standards for DCs contacted with various pathogens are generated and used in subsequent patient assays to determine contact and qualify immunological responses to pathogens.

Gene expression analysis : DCs and macrophages / monocytes from various donors are cultured in the presence and absence of pathogens to initiate a response to pathogens and then harvested. Total RNA is extracted from uninfected and infected cells and used to create a reference array. Alternatively, total RNA is converted to cDNA before creating the reference array. Patient-derived DC samples are collected, nucleic acids are extracted, hybridized to the microarray, and then scanned. In microarray data processing, we use filtering methods based on constant changes in mean difference intensity values. For raw data analysis, we use GenNet, an extremely robust expression data analysis platform. The use of such a platform meets high throughput requirements and is scalable.

Pathogen genotyping : Since DCs and macrophages serve as pathogen reservoirs, the enrichment of these cells and the use of genotyping for the presence of pathogens provides a novel screening method. Conventional diagnostic methods for viral and bacterial diseases require, for example, detection of the pathogen itself by blood culture or detection of pathogen-specific proteins or DNA / RNA by, for example, PCR and correlation of these findings with clinical symptoms. In order to overcome the limitations associated with traditional screening methods, we have developed high-throughput genotype assays and use them to screen the majority of pathogens. These assays can also detect molecular mutations in microbial strains; thus allowing for identification of infection routes, origins and relationships such as, for example, specific bacterial, viral, myocardial or parasitic strains.

Mass Spectrometry (MS) : To improve peptide identification success and productivity, we used SELDI and MALDI time-of-flight (TOF) mass spectrometers. These are the most commonly used mass spectrometry methods for detecting peptide mass fingerprints. This MALDI-O-TOF system uses orthogonal injection to introduce sample ions from a MALDI source into a reflection TOF mass spectrometer. The conventional MALDI-TOF system (straight or reflection mode) MALDI is directly connected to the TOFMS. This direct connection affects the accuracy, resolution and sensitivity of the instrument as any discrepancies associated with the sample target are carried to the detector. On the other hand, using orthogonal geometry, the MALDI source is separated from the TOF, thus eliminating discrepancies, increasing performance, and simplifying method development. Establish the protein signature found in the untreated culture compared to the treated culture.

Biomarker detection : To detect biomarkers, we use commercially available pattern recognition to discover software from Eclipse Diagnostics or similar software. This software allows for rapid detection of genomic and proteomic biomarkers and other complex biological relationships. These biomarkers are part of the database and are used as pathogen-specific references.

Advantages First, bone marrow cells concentrate pathogens within the cells; thus, improved detection sensitivity. Second, cDNA microarray technology is a high-resolution technique that can analyze more than 52,000 genes per sample and is independent of culturing pathogens from blood. Our microarray construction method involves a long 70mer probe design and 30-fold internal redundancy per gene. Third, the ability to diagnose before symptoms occur. In the case of genetically engineered pathogens, conventional molecular biology, ELISA or PCR-based techniques must be created to detect new pathogens. By assessing major cellular pathways (eg, apoptosis, inflammation, NFκB and inflammation mediators), we can detect early events of contact. We have developed a method (1.5 hours) that rapidly concentrates DCs and macrophages from blood that does not require the use of expensive cytokines to drive long-term culture or differentiation. On the other hand, current methods require culturing DC precursors for 7 days. DCs in culture upon prolonged contact with cytokines are known to induce specific changes in cellular pathways and have been demonstrated to alter pathogen infectivity, gene activation and protein synthesis. High-throughput (HT) gene arrays allow analysis of over 1,000 samples per day. The number of samples processed per day is device dependent and is the same as in proteomic technology (eg 10,000 samples per day). Integration of the HT system with genomic and proteomic databases improves detection efficiency and enables real-time monitoring of disease progression. Collectively, the combination provides a high complexity genomic and proteomics-based array and information database for hospitals and laboratories that can be shared in real time on the network.

  The present invention provides a method for rapidly identifying in vivo and environmental pathogens. The following pathogens can be detected and characterized using APC and the techniques described herein: bacteria, viruses, myocardium, prions and protozoa.

  Representative bacteria that are presented to APCs to create pathogen-contacting APC signatures include Staphylococcus, such as S. epidermis and S. aureus; Micrococcus; S. pyogenes, S. equis, S. zooepidemicus, S. equisimilis, S. pneumoniae and S. agalactier Streptococcus such as agalactiae; Corynebacterium such as C. pyogenes and C. pseudotuberculosis; Elidiperos such as E. rhusiopathiae Erysipelothrix; Listeria such as L. monocytogenes; B. Bacillus such as B. anthracis; Clos such as C. perfringens Torijiumu (Clostridium); as well as tuberculosis (M. tuberculosis) and M. leprae (M. leprae) Gram-positive and Gram-negative bacteria such as Mycobacterium (Mycobacterium) and the like. Gram-negative bacterial species include Escherichia such as E. coli O157; Salmonella such as S. typhi and S. gallinarum; S. disenterier (S. Shigella such as dysenteriae; Vibrio such as V. cholerae; Yersinia such as Y. pestis and Y. enterocolitica; Proteus such as P. mirabilis; Bordetella such as B. bronchiseptica; Pseudomonas such as P. aeroginosa; K. pneumoniae; Klebsiella such as Pneumoniae; Pasteurella such as P. multocida; Moraxella such as M. bovis; S. marcescens Serratia; H. influenza, etc. Fils (Hemophilus) and is exemplified by species including Campylobacter (Campylobacter) species, but are not limited to. Other species suitable for the assay of the present invention include spirochetes such as those causing Lyme disease, Enterococcus, Neisseria, Mycoplasma, Chlamia, Francisella, Pasteurella ), Brucella and Enterobacteriaceae. CDC biological pathogens A, B and C biological pathogens can also be detected. Yet another example of a pathogenic bacterial species that can be detected according to the present invention is the standard taxonomic and explanatory such as Bergey's Manual of Determinative Bacteriology), 9th edition, 1994, Williams and Wilkins, Baltimore, Md. Can be obtained by referring to other studies.

  Representative viruses presented to APC to make pathogen contact APC signatures include adenoviruses (such as those that can be found in pediatric gastroenteritis, acute hemorrhagic cystitis, nonbacterial pneumonia and viral conjunctivitis) , Herpesviruses (type I and type II herpes simplex, varicella-zoster (causes of blisters), cytomegalovirus and mononucleosis (causative Epstein-Barr virus), poxviruses (such as smallpox) Causative organisms (such as Variola major and Variola minor), hepatitis A, B and C, vaccinia virus, hantavirus and infectious molluscum), picornavirus (rhinovirus ( Cold, which is also caused by a coronavirus) Enza and respiratory syncytial virus (RS)), parainfluenza virus (including diseases such as epidemic parotitis) and measles, rhabdovirus (rabies), vesicular stomatitis (VSV) , Togaviruses such as Rubera-Flaviviruses such as causative bacteria that cause only three days and encephalitis (EEE, WEE and VEE), dengue fever, West Nile fever, yellow fever and encephalitis Included are papovavirus infections such as papillomaviruses such as bunyavirus and arenavirus, reovirus, SARS, coronaviruses that cause hepatitis, and retrovirus infections such as HIV, HTLV-1 and HTLV-II.

  Representative fungi that are presented to APCs to generate pathogen contact APC signatures include, for example, Candida such as C. albicans; Cryptococcus such as C. neoformans; Malassezia ( Malassezia (Pityrosporum); H. histoplasma such as H. capsulatum; C. immitis such as C. immitis; H. destruens; Hytrumyces such as destruens; Blastomyces such as B. dermatiditis; Aspergillus such as A. fumigatus; P. marneffei Penicillium; Pseudallescheria; Fusarium; Paecilomyces; Mucor / Rhizopus; and P. Cali Includes species; (P. carinii) Pneumocystis, such as (Pneumocystis). Subcutaneous fungi such as Rhinosporidium and Sporothrix, and dermatophytes such as Microsporum and Trichophyton species can be prevented and treated herein according to embodiments of the invention It is. Other fungi that can be detected include Trichophyton; Microsporum; Epidermophyton; Basidiobolus; Conidiobolus; Rhizopus kuningamelia (Rhizopus Cunninghamelia); Rhizomucor; Paracoccidioides; Pseudallescheria; Rhinosporidium and Sporothrix.

  Representative protozoa that are presented to APCs to create pathogen-contacting APC signatures include 1 amoeba, ciliate, flagella neglect and spore, such as Plasmodium, Trypanosoma or Cryptosporidium One or more single cells, usually microscopic sized eukaryotes.

Example 2
Generation of antigen-presenting cell (APC) specific signature is a two-step method. The first stage involves obtaining an immune cell population, fractionating the cell membrane, and enriching the protein from the membrane, cytoplasm and nucleus. The preferred cell is the bone marrow lineage, but PBMC is preferred. Methods for enriching bone marrow cell populations containing DC are described in US Pat. Nos. 6,589,526 and 6,194,204. Bone marrow cells comprise approximately 90% to 10% of the population of monocytes and dendritic cells. Monocyte antigen markers include CD14 +, HLA-DR or MHC class II, CD80 + CD86 +. Antigen markers for DC include CD2 +, CD5 +, CD14 + CD83 + and CD90 +. These are obtained by a positive or negative selection method. A preferred cell type is bone marrow that expresses antigenic markers consistent with DC and monocyte cells. At present, it is preferred to use immediately after isolation, ie blood purified bone marrow cells, rather than cultured bone marrow cells.

The following is a suggested procedure for isolating monocytes from PBMC: buffy coats were isolated from healthy volunteers (Transfusion Therapy, Children's Hospital, Boston, Mass.), Washed with PBS and concentrated. The buffy coat concentrate was then incubated with an improved monocyte enrichment means such as, for example, the RosetteSep kit commercially available from StemCell Technologies. This rosette cocktail contains anti-CD3, anti-CD19, anti-CD54 and anti-CD62 monoclonal antibodies that bind to T cells, B cells, NK cells and granulocytes. After a 30 minute incubation, the population was stratified with a Ficoll gradient and centrifuged at 2500 rpm for 30 minutes (Sorvall RT 6000, DuPont, Wilmington, DE) from high density (T, B, granulocytes and NK cells). Low density DC and Mo were separated. The low density cell population was> 95% CD14 ^high by flow cytometry. These cells were incubated with mouse mAb (ascites) diluted 100-fold against human CD2 for 30 minutes at 4 ° C (1O1d2-4Cl (anti-Tl12); Dana-Farber Cancer Institute, Boston, MA) (26), washed And incubated with goat anti-mouse IgG magnetic beads (Miltenyi Biotech). Following incubation, the preparation was passed through a magnetic column according to the manufacturer's instructions. The magnetic column retained CD2 + cells and was> 96% pure, whereas CD2 cells were> 95% pure by flow cytometry using anti-CD2 and anti-CD14. Magnesium and calcium free HBSS (Cellgro; Fisher Scientific, Pittsburgh, PA) with 10% v / v heat inactivated pool human serum (PHS) (Nabi, Boca Raton, FL) and human IgG (50 mg / ml; Immuno Block buffer containing AG, Vienna, Austria) was used to prevent non-specific mAb binding during each step of isolation or flow cytometry analysis. For morphological and functional studies of CD2 + and CD2-Mo incubated with noncytokine immediately after isolation, we have 10% heat inactivated PHS, 20 μg / ml gentamicin, 100 U / Culture medium (CM) containing RPMI 1640 (Cellgro) supplemented with ml penicillin and 100 μg / ml streptomycin was used (Life Technologies, Gaithersburg, MD).

  The second step involves obtaining a mass spectrum from DC using, for example, QSTAR (ABI), Ciphergen SELDI TOF or Perkin Elmer proTOF. Spectra were taken for naive APCs and those contacted with pathogens, tumors or other antigens. In this example, we obtain individual data sets (signatures) from pathogen-APC or pathogen-food samples (i.e., Listeria-APC, Listeria-milk) to produce bacterial contaminated milk. And a method for producing a profile of uncontaminated milk. It also provides signatures of Listeria infected individuals obtained from individual APC sampling.

  Each profile is measured by properties such as m / z size (kD), m / z intensity (uAmp) and standard deviation values, the cell membrane, cytoplasmic and nucleoprotein characteristics, protein charge (i.e. Positive and negative), Cu2 + chelate properties, cleavage patterns of native or denatured proteins by various endopeptidases, and the like. The frequency of occurrence identifying signature features is supported by obtaining spectra of repetitive samples, preferably 3-4 samples, thereby providing a consensus signature.

  Some commonly used methods for isolating, fractionating or concentrating sample proteins are as follows, others are known in the art. PARIS Kit-This is an Ambion kit that allows rapid isolation of RNA and proteins from a sample. This kit can be used for studies involving the isolation of APC proteins from APC virus or bacterial co-cultures. For more sensitive detection and characterization of samples, it is advantageous to use cell membrane fractions obtained by common molecular biological techniques. The combination of membrane, cytoplasm and nucleoprotein enhances the sensitivity of APC-based detection methods.

  Bacterial lysis by sonication can be used for gram-negative and gram-positive bacteria, using sonication and optionally being strongly advanced for gram-negative bacteria or microbial profiles with thick or robust cell membranes , Tween, Triton X-100, digitonin, CHAPS, SDS, nonidet, and other surfactants are used. This protocol was used for the milk study shown in FIGS. Bacteria are recovered from the agar plate in 5 ml TEN buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 100 mM NaCl). Pellet bacteria at 5,000 xg for 10 minutes. Samples are subjected to 3 rounds of freeze / thaw in a dry ice / ethanol bath and thawed at 37 ° C. The bacterial pellet is resuspended in a small volume (0.1-0.5 ml) of ice-cold MTBS buffer (16 mM Na2HPO4, 4 mM NaH2P04, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF). The suspension is sonicated in 15 second bursts and then incubated for 30 seconds on ice (4 rounds of sonication). Pellet the bacterial pieces at 14,000 rpm. Remove the supernatant to a new tube. Measure protein concentration by Bradford protein assay, standard curve, UV absorption or other methods. Store at -70 ° C until SELDI analysis.

  Repeated freeze-thawing involves repeating the freeze-thaw cycle to shear the cells. Samples were frozen in a dry ice-ethanol mixture and thawed at 37 ° C. These steps were repeated 4 times and the sample was centrifuged at 14,000 rpm for 5 minutes in a microcentrifuge to form a clear supernatant. The supernatant was removed and stored at -70 ° C. until sample manipulation. Bacterial lysis by French Press can be used for Gram negative bacteria and is not recommended for Gram positive bacteria. To carry out this technique, the bacterial pellet is resuspended in 20 mM HEPES pH 7.4, 50 mM NaCl, 1% Triton-X 100, 1 mM PMSF. Separate using a French press at 750 psi. Cell debris is removed by centrifugation at 20,000 xg for 20 minutes at 4 ° C. Measure protein concentration in the supernatant by Bradford protein assay or similar assay. Store aliquots at -70 ° C until SELDI technique is performed. BugBuster Extraction Kit—This is a commercial kit sold by Novagen that can gently break the cell wall of E. coli to release active protein. This is a simple, quick and low cost alternative to French press or sonication to release expressed target protein into cell preparations. Alternatively, buffers such as 10 mM Tris-HCl pH 7.4, 8 M Urea, 2% (w / v) CHAPS, ImM PMSF can also be used as the lysis buffer. Store aliquots at -70 ° C until SELDI technique is performed.

  The SELDI experimental protocol described below uses an IMAC ProteinChip Array (PCA). The IMAC array is coated with NTA functional groups to capture the transition metal for subsequent metal affinity binding proteins. In these profiling studies, the array is charged with copper before the sample is applied to the surface. Selectively by imidazole concentration in binding buffer. Increasing the imidazole concentration in the binding / wash buffer reduces the binding of proteins with low affinity for the metal and lowers the background signal. The protocol for IMAC PCA is described in detail below and is similar to other Ciphergen PCA protocols.

  Using an 8-spot array: Assemble the PCA on the bioprocessor and add 50 microliters of IMAC additive to each well. Vortex for 5 minutes at room temperature. Remove buffer from the wells. Rinse with water. Add 50 microliters of IMAC neutralization buffer to each well. Vortex for 5 minutes at room temperature. Remove buffer from well and rinse. Add 150 microliters of IMAC binding buffer to each well and vortex for 5 minutes at room temperature. Remove buffer. Repeat the washing step with binding buffer twice. Next, 90 microliters of IMAC binding buffer and 10 microliters of sample are added and vortexed for 30 minutes at room temperature. The ratio of IMAC binding buffer to sample concentration may vary depending on the desired protein concentration. The sample is removed and washed 3 times with IMAC binding buffer, each wash requiring a 5 minute stirring step. When finished, rinse with deionized water and drain the bioprocessor wells and allow to air dry. Apply 1.0 microliter of EAM (matrix) solution to each spot and allow to air dry. Analyze PCA with Ciphergen Chip Reader.

  On the other hand, the Perkin-Elmer proTOF experimental protocol directly applies a protein sample isolated on a MALDI surface. However, in this method, the MALDI surface binds to everything in the sample (ie protein and non-protein). Cleanup samples, for example, Millipore ZIP TIPS®, filtered ion exchange pipettes that can remove certain proteins, thereby reducing total protein levels and complexity of sample mixtures Manufacturers suggest microscale protein purification using chips. However, this technique is sensitive enough that it can affect the signature that results in different chip performance from chip to chip.

  The next step involves the derivatization of the marker protein from the pathogen-APC interaction. Once the marker protein is isolated, the protein provides a template for making diagnostic antibodies. These antibodies against derivatized APC proteins can be attached to a multiplexer or fluorescent reader for diagnostic purposes, for example, and used in immunological assays.

Example 3
This experiment examined the reproducibility of DC APC-derived signatures contacted with bacterial and viral pathogens. Listeria monocytogenes were processed by the bacterial lysis and sonication methods described above and evaluated for metal binding (FIGS. 1-2) or hydrophobic surfaces (FIGS. 3-4). Samples were processed in parallel and analyzed with different PCA chips in different chip analyzers. The results demonstrate excellent experimental reproducibility. Data are shown as spectra (Figures 1, 3) or gel (Figures 2, 4) images.

  FIG. 5 shows untreated / uninfected bone marrow cells (Mx, upper panel, (DC) mixture of dendritic cells, middle panel and (Mo) monocytes, lower panel), DC and Mo cytoplasmic protein profiles ( Signature). Differences are observed in the individual profiles of DC and Mo.

  FIGS. 6-11 show Mx, Mo and DC cytoplasmic proteins cultured in the presence of control adenovirus (FIGS. 6, 8 and 10) or in the presence of single gene replacement adenovirus (FIGS. 7, 9 and 11). Illustrate the profile (signature). The observed signature shows that APCs infected with wild type and mutant adenoviruses can be identified and distinguished using the present invention. Unique protein signatures can be obtained that can identify viruses with a single gene replacement / modification.

  Figures 12-14 show protein profiles obtained from nucleoprotein extracts of Mx (Figure 12), DC (Figure 13) and Mo (Figure 14) co-cultured in the presence of Listeria monocytogenes. Signature). APC cultured in the presence of other Gram positive and Gram negative bacteria also creates a unique signature that can be used to identify microorganisms co-cultured with APC. Listeria alone, a proteomic signature that does not grow with APC (bottom panel in Figure 15), is distinct from the three co-culture signatures, suggesting that Listeria overgrowth does not occur and thereby does not contaminate APC co-culture. Yes.

  FIGS. 16-17 illustrate the spectral profiles (signatures) of skim milk or whole milk mixed with Listeria. FIG. 15 shows the resulting spectral image, while FIGS. 16-17 show gel images. The results demonstrate the ability of the present invention to detect the presence of unique pathogens in milk and other food products, regardless of the APC detection method.

Example 4
In addition to pathogenic bacteria, the diagnostic method of the present invention includes detection, diagnosis and staging of various cancers and genetic diseases. Cancer can be detected by a number of markers. Malignant cells often express antigens that are not found in normal cells: some of these antigens are found on the cell surface, eg CEA (carcinoembryonic antigen) and differentially glycosylated (low Glycosylation) MUC-1 is two well-known tumor-associated antigens. MUC-1 / DF-3 is overexpressed, among other things, in the majority of human cancers, multiple myeloma, acute myeloid leukemia, acute lymphocytic leukemia and follicular lymphoma. The antigen can initiate an HLA-restricted T cell response by presentation of the antigen by DC (see Brossart et al., (2001) Cancer Res. Sept.:61(18):6846-50). Other proteins that are upregulated in cancer cells include vascular endothelial growth factor (VEGF), Her-2 / neu and hepsin. Cell adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and E-selectin (ELAM-1) are important in a complex series of events related to inflammatory responses associated with cancer and tumor suppression Play an important role.

  In addition to pathogenic bacteria, the diagnostic methods of the present invention include detection, diagnosis and staging of various tumors and malignant neoplasms. The present invention can detect any of a variety of malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to infiltrate surrounding tissues and metastasize to new biological sites. For example, APC is cultured with the following cancers to produce APC signatures of cancer contact cells: astrocytoma, glioma, ependymoma, osteosarcoma, Ewing sarcoma, retinoblastoma, bladder cancer, small and non-small Cell lung cancer, barley cell lung cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, vaginal cancer, ovarian cancer, liver cancer, acute lymphocytic leukemia, acute myeloid leukemia, lymphoma, myeloma, basal cell carcinoma , Melanoma, follicular thyroid cancer, bladder cancer, glioma, myelodysplastic syndrome, testicular cancer, gastric cancer, esophageal cancer, laryngeal cancer, squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, urothelial cancer, breast cancer or prostate cancer.

  APC, particularly CD2 + DC, is cultured with primary or metastatic tumor cells obtained by biopsy, preferably with cells taken from a typical stage of tumor growth. Alternatively, DCs are cultured with purified CEA (chorioembryonic antigen) preparations or differentially glycosylated (hypoglycosylated) MUC-1 or other tumor associated antigens. DC lysates are used to make cDNA, which is then used to make DC reference standard arrays for high-throughput screening. An array is created for each major species listed above, and preferably includes each stage of a particular cancer type.

Example 5
Develop kits to isolate samples from subjects and the environment.

  For patient diagnostic applications, the kit concentrates reagents and materials for obtaining and isolating blood samples from patients, and cells such as APC, PBMC, preferably DC, and processes the cells to cytoplasm , Including reagents and materials to form nuclei or membrane fractions and optionally process large proteins into small peptides.

  The kit may further comprise suitable chips or plates and reagents for use in mass spectrometry, such as those made by Ciphergen for SELDI and Perkin Elmer for MALDI-O-TOF. The kit also includes suitable instructions for use. In certain embodiments, the kit is an APC array as described above, eg, for use in diagnosing and staging cancer, or for determining the infection and progression of infection, or for forensic analysis. Contains one or more. Other kit components include controls such as reference proteins used to calibrate the mass spectrometer. In yet other embodiments, the kit comprises albumin or a high molecular weight protein and is used to enhance the resolution of low molecular weight proteins in the signature (albumin bump technique). The kit used for the patient is suitable for human and veterinary use.

  The following kits are provided herein: biological weapon defense kits for identifying pathogens associated with biological weapons in environmental and contact subjects; agricultural kits for sampling food dairy and livestock contamination; Endocrine and metabolic kits for assessing the endocrine and metabolic function of subjects; neurological kits for assessing degenerative changes; infectious disease kits for identifying pathogens in contact subjects; for assessing fetal health Cancer kit for diagnosing, staging, monitoring chemotherapy and disease progression in a subject; cardiovascular system for detecting early signs of heart damage and ischemia and vascular occlusion Kit; ie, a renal kit for detecting a subject's damage due to contrast agents and chemotherapeutic agents.

Equivalents From the above detailed description of specific embodiments of the invention, it should be apparent that unique detection methods have been described. Although certain aspects are disclosed in detail herein, this is done by way of example only for purposes of illustration and is not intended to be limiting with respect to the following appended claims. In particular, it is contemplated that various substitutions, changes and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. For example, the selection of spectra or APC used in the detection method will be routine for those skilled in the art having knowledge of the embodiments described herein.

References

It is the figure which processed the Listeria monocytogenes by the bacteria lysis and the ultrasonic treatment method, and evaluated the metal bond. Data are shown as spectral images. It is the figure which processed the Listeria monocytogenes by the bacteria lysis and the ultrasonic treatment method, and evaluated the metal bond. Data are shown as gel images. It is the figure which processed the Listeria monocytogenes by the bacteria lysis and the ultrasonic treatment method, and evaluated about the hydrophobic surface. Data are shown as spectral images. It is the figure which processed the Listeria monocytogenes by the bacteria lysis and the ultrasonic treatment method, and evaluated about the hydrophobic surface. Data are shown as gel images. Illustrate untreated / uninfected bone marrow cells (Mx, upper panel, (DC) mixture of dendritic cells, middle panel and (Mo) monocytes, lower panel), DC and Mo cytoplasmic protein profiles (signature) It is a figure to do. FIG. 3 illustrates the cytoplasmic protein profile (signature) of Mx cultured in the presence of a control adenovirus. It is a figure which illustrates the cytoplasmic protein profile (signature) of Mx cultured in the presence of a single gene-replaced adenovirus. FIG. 2 illustrates the cytoplasmic protein profile (signature) of Mo cultured in the presence of a control adenovirus. FIG. 6 illustrates the cytoplasmic protein profile (signature) of Mo cultured in the presence of a single gene-replaced adenovirus. FIG. 2 illustrates the cytoplasmic protein profile (signature) of DC cultured in the presence of a control adenovirus. It is a figure which illustrates the cytoplasmic protein profile (signature) of DC cultured in presence of the adenovirus substituted with the single gene. It is a figure which illustrates the protein profile (signature) obtained from the nucleoprotein extract of Mx cocultured in the presence of Listeria monocytogenes. It is a figure which illustrates the protein profile (signature) obtained from the nuclear protein extract of DC co-cultured in presence of Listeria monocytogenes. It is a figure which illustrates the protein profile (signature) obtained from the nuclear protein extract of Mo co-cultured in presence of Listeria monocytogenes. It is a figure which illustrates the spectral profile (signature) of skim milk mixed with Listeria or whole milk. The resulting spectral image is shown. It is a figure which illustrates the spectral profile (signature) of skim milk mixed with Listeria or whole milk. A gel image is shown. It is a figure which illustrates the spectral profile (signature) of skim milk mixed with Listeria or whole milk. A gel image is shown.

Claims (29)

obtaining a blood sample having a sub-population of antigen-presenting cells from a mammal;
b. substantially isolating antigen-presenting cells from a blood sample;
c. inducing the genome or proteomics mammalian sample signature of the isolated antigen presenting cell, wherein the mammalian sample signature indicates the metabolic state of the antigen presenting cell in the mammal;
d. deriving one or more genomic or proteomic reference signatures of antigen presenting cells from a reference subject having a disease state; and
e. A diagnostic method comprising the step of comparing a mammalian sample signature with a reference signature, wherein the match between the mammalian sample signature and the reference signature indicates the presence of a disease state in the mammal.   2. The diagnostic method according to claim 1, wherein the antigen-presenting cell is a dendritic cell.   2. The diagnostic method according to claim 1, wherein the disease state is cancer or a cell proliferative disease.   2. The diagnostic method according to claim 1, wherein the disease state is a pathogen infection.   5. The diagnostic method according to claim 4, wherein the pathogen infection is a viral infection.   5. The diagnostic method according to claim 4, wherein the pathogen infection is a bacterial infection.   An array comprising a plurality of addresses, wherein a nucleic acid sample corresponding to a gene expressed by an antigen-presenting cell is added to each address.   8. The array of claim 7, further comprising a plurality of secondary addresses, wherein each secondary address is appended with a nucleic acid sample corresponding to a gene expressed by an antigen presenting cell experienced in encountering the antigen.   9. The array according to claim 8, wherein the antigen-presenting cell is a dendritic cell.   10. The array of claim 9, wherein the antigen is a cancer antigen.   10. An array according to claim 9, wherein the antigen is a viral antigen.   10. An array according to claim 9, wherein the antigen is a bacterial antigen. obtaining an isolated antigen-presenting cell population;
b. culturing antigen presenting cells in the presence of a foodborne pathogen, thereby creating a reference cell having a proteomic or genomic reference signature specific for the foodborne pathogen and obtaining a reference signature;
c. obtaining a food sample;
d. culturing naive antigen-presenting cells with the sample food;
e. obtaining a sample signature from the cultured antigen-presenting cells; and
f. A diagnostic method comprising the step of comparing the sample signature with a reference signature, wherein the consistency between the sample signature and the reference signature indicates the presence of a foodborne pathogen in the food.   14. The method of claim 13, wherein the antigen presenting cell is a dendritic cell.   14. The method of claim 13, wherein the foodborne pathogen is a bacterial pathogen.   14. The method of claim 13, wherein the foodborne pathogen is a viral pathogen. obtaining a blood sample having a subpopulation of antigen presenting cells from a mammalian patient undergoing treatment for the disease;
b. substantially isolating antigen-presenting cells from a blood sample;
c. inducing a genomic or proteomic patient signature for the isolated antigen presenting cell, wherein the patient signature indicates the metabolic state of the antigen presenting cell in the subject;
d. deriving one or more genomic or proteomic reference signatures of antigen presenting cells from a reference subject having the same disease as the patient; and
e. A diagnostic method comprising the step of comparing a patient signature with a reference signature, wherein the consistency between the patient signature and the reference signature is reduced during treatment, thereby indicating the effectiveness of the treatment in treating the disease.   18. The method of claim 17, wherein the disease is a cell proliferative disease and the treatment is administration of an antineoplastic agent.   18. The method of claim 17, wherein the disease is a cell proliferative disease and the treatment elicits an immune response against the cell proliferative disease.   18. The method of claim 17, wherein the disease is a bacterial infection and the treatment is administration of an antibacterial agent.   18. The method of claim 17, wherein the disease is a viral infection and the treatment is administration of an antiviral agent. obtaining a blood sample having a subpopulation of antigen-presenting cells from a diseased mammal;
b. substantially isolating antigen-presenting cells from a blood sample;
c. deriving one or more marker polypeptides from the antigen-presenting cell, wherein the marker polypeptide is expressed in the antigen-presenting cell in response to the antigen contact and the contacted antigen is associated with the disease or of the disease Causal factor, stage,
d. obtaining an antibody against the marker polypeptide; and
e. A diagnostic method comprising the step of detecting the presence or absence of a polypeptide that binds to an antibody in an antigen-presenting cell of a subject, wherein the presence of the polypeptide confirms the presence of a disease in the subject.   The diagnostic method according to claim 22, wherein the antigen-presenting cell is a dendritic cell.   23. The diagnostic method according to claim 22, wherein the disease is cancer or a cell proliferative disease.   23. The diagnostic method according to claim 22, wherein the disease is a pathogen infection.   23. The diagnostic method according to claim 22, wherein the disease is a viral infection.   23. The diagnostic method according to claim 22, wherein the disease is a bacterial infection.   23. The diagnostic method according to claim 22, wherein the disease is prion infection.   23. The diagnostic method according to claim 22, wherein the disease is a fungal infection.

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