IMMUNE CELL BIOSENSORS AND METHODS OF USING SAME

FIELD OF THE INVENTION

[0001] The present invention relates to immunological cells that are useful in detecting changes in physiological states, which provide for methods of diagnosing diseases or monitoring the course of patient therapy.

BACKGROUND

[0002] In many diseases such as cancer, autoimmune diseases or cardiovascular disorders peptides of normal or abnormal cellular proteins are presented on the cell surface which can not be found on the cell surface of healthy individuals. Inadequate antigen presentation in humans results in the failure of human immune system to control and clear many pathogenic infections and malignant cell growth. Successful therapeutic vaccines and immunotherapies for chronic infection and cancer rely on the development of new approaches for efficient antigen presentation to induce a vigorous immune response which is capable of controlling and clearing the offensive antigens.

[0003] The ability of T cells to recognize an antigen is dependent on association of the antigen with either MHC Class I (MHC-I) or Class II (MHC-II) proteins. For example, cytotoxic T cells respond to an antigen in association with MHC-I proteins. Thus, a cytotoxic T cell that kills a virus-infected cell will not kill a cell infected with the same virus if the cell does not also express the appropriate MHC-I protein. Helper T cells recognize MHC-II proteins. Helper T cell activity depends in general on both the recognition of the antigen on antigen presenting cells and the presence on these cells of "self MHC-II proteins. This requirement to recognize an antigen in association with a self-MHC protein is called MHC restriction. MHC-I proteins are found on the surface of virtually all nucleated cells. MHC-II proteins are found on the surface of certain cells including macrophages, B cells, and dendritic cells (DCs) of the spleen and Langerhans cells of the skin.

[0004] A crucial step in mounting an immune response in mammals, is the activation of CD4+ helper T-cells that recognize major histocompatibility complexes (MHC)-II restricted exogenous antigens. These antigens are captured and processed in the

cellular endosomal pathway in antigen presenting cells, such as dendritic cells. In the endosome and lysosome, the antigen is processed into small antigenic peptides that are presented onto the MHC-II in the Golgi compartment to form an antigen-MHC-II complex. This complex is expressed on the cell surface, which expression induces the activation of CD4+ T cells.

[0005] Other crucial events in the induction of an effective immune response in an animal involve the activation of CD8+ T-cells and B cells. CD8+ cells are activated when the desired protein is routed through the cell in such a manner so as to be presented on the cell surface as processed proteins, which are complexed with MHC-I antigens. B cells can interact with the antigen via their surface immunoglobulins (IgM and IgD) without the need for MHC proteins. However, the activation of the CD4+ T-cells stimulates all arms of the immune system. Upon activation, CD4+ T- cells (helper T cells) produce interleukins. These interleukins help activate the other arms of the immune system. For example, helper T cells produce interleukin-4 (IL-4) and interleukin-5 (IL-5), which help B cells produce antibodies; interleukin-2 (IL-2), which activates CD4+ and CD8+ T-cells; and gamma interferon, which activates macrophages. Since 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, the initial event of activating the helper T cells in response to an antigen is crucial for the induction of an effective immune response directed against that antigen.

[0006] Peptides and proteins expressed in diseased cells can be used as markers for the identification of such abnormal cells. Furthermore, the detection of antibodies in serum or other body fluids directed to these peptides or proteins can also be used as indicator of risk or as prognostic indicator. However, the concentrations of these disease related peptides are quite low, and isolating and identifying them is usually only efficacious when the disease predominates in the individual, which by that time, usually precludes effective treatment. There remains a need in the art for a rapid and sensitive assay for detection of a pathological state in a mammal.

SUMMARY OF THE INVENTION

[0007] The present invention is based on the plasticity of antigen presenting cells, and the highly specific metabolic changes APCs, particularly DCs undergo after they

encounter antigens. These changes can be quantitated and when compared to reference positive (antigen exposed) and negative (naϊve) controls of APCs, provide information about the immune state and microenvironments of the mammal from which they are obtained.

[0008] In one aspect, the invention provides a diagnostic method having the steps of obtaining from a mammalian subject a sample of blood having a subpopulation of antigen presenting cells, substantially isolating the antigen presenting cells from the blood sample, deriving a genomic or proteomic mammalian subject signature for the isolated antigen presenting cells wherein the mammalian subject signature indicates the metabolic state of the antigen presenting cells in the subject, deriving one or more genomic or proteomic reference signatures of antigen presenting cells from a reference subject having a disease state, and comparing the mammalian subject signature to the reference signature, wherein congruity between the mammalian subject signature and the reference signature indicates the presence of the disease state in the mammal. A mammalian subject is preferably a human, but can also be a veterinary subject such as a dog, cat, horse, pig, sheep, goat, or other mammal. In one embodiment, the antigen presenting cells are dendritic cells. In another embodiment, the disease state is a cancer or cell proliferative disorder. In yet another embodiment, the disease state is a pathogenic infection. In still another embodiment, the pathogenic infection is a viral infection. In yet still another embodiment, the pathogenic infection is a bacterial infection.

[0009] In another aspect, the invention provides an array having a plurality of addresses, each address having affixed thereto a sample of nucleic acid corresponding to genes expressed by an antigen presenting cell. In one embodiment, the array further includes a plurality of secondary addresses, each secondary address having affixed thereto a sample of nucleic acid corresponding to genes expressed by an antigen presenting cell that has encountered an antigen. In another embodiment, the antigen presenting cell is a dendritic cell. In yet another embodiment, the antigen is a cancer antigen. In still another embodiment, the antigen is a viral antigen. In even another embodiment, the antigen is a bacterial antigen. In still another embodiment, the antigen is a fungal antigen.

[00010] In still another aspect, the invention provides a diagnostic method including the steps of obtaining a population of isolated antigen presenting cells, culturing the antigen presenting cells in the presence of a food-borne pathogen thereby producing reference cells, the reference cells having a proteomic or genomic reference signature specific for the food-borne pathogen, and obtaining the reference signature obtaining a sample of a food product, culturing naϊve antigen presenting cells with the sample food product, obtaining a sample signature from the cocultured antigen presenting cells, and comparing the sample signature to the reference signature, wherein congruity between the sample signature and the reference signature indicates the presence of the food-borne pathogen in the food product. In one embodiment, the antigen presenting cell is a dendritic cell. In another embodiment, the food-borne pathogen is a bacterial pathogen. In still another embodiment, the food-borne pathogen is a viral pathogen. In still another embodiment, the food-borne pathogen is a prion pathogen. In a related aspect, the invention provides for obtaining the antigen presenting cells from a livestock mammal, and assaying for APC exposure to a food-borne pathogen in the livestock mammal, and the consequent gene and protein expression changes in the APC that follow from antigen contact. In one embodiment, the antigen presenting cell is a dendritic cell. In another embodiment, the food-borne pathogen is a bacterial pathogen. In still another embodiment, the food-borne pathogen is a viral pathogen. In still another embodiment, the food-borne pathogen is a prion pathogen, for example, the prion that causes Bovine Spongiform Encephalopathy (BSE).

[00011] In even yet another aspect, the invention provides a diagnostic method including the steps of obtaining from a patient being treated for a disorder, a sample of blood having a subpopulation of antigen presenting cells, substantially isolating the antigen presenting cells from the blood sample, deriving a genomic or proteomic patient signature for the isolated antigen presenting cells wherein the patient signature indicates the metabolic state of the antigen presenting cells in the subject, deriving one or more genomic or proteomic reference signatures of antigen presenting cells from a reference subject having the same disorder as the patient, and comparing the patient signature to the reference signature, wherein the congruity between the patient signature and the reference signature decreases during the treatment, thereby indicating the efficacy of the treatment in treating the disorder. In one embodiment,

the disorder is a cell proliferative disease or a cancer, and the treatment is administration of an antineoplastic agent. In another embodiment, the disorder is a cell proliferative disease or a cancer, and the treatment provokes an immune response against the cell proliferative disease. In yet another embodiment, the disorder is an autoimmune disease, and the treatment reduces the autoimmune response. In still another embodiment, the disorder is a bacterial infection, and the treatment is administration of an antibacterial agent. In even another embodiment, the disorder is a viral infection, and the treatment is administration of an antiviral agent. In even still another embodiment, the disorder is a fungal infection and the treatment is administration of an antifungal agent. In also another embodiment, the disorder is a genetic disorder, and the treatment is gene replacement therapy.

[00012] In another aspect, the invention provides for a antigen presenting cell, wherein the cell has been cultured in the presence of an antigen, and wherein the antigen expresses a plurality of genes that are specifically upregulated in response to antigenic challenge. In one embodiment, the antigen presenting cell is a dendritic cell. In yet another embodiment, the antigen is a cancer antigen. In still another embodiment, the antigen is a viral antigen. In even another embodiment, the antigen is a bacterial antigen. In still another embodiment, the antigen is a fungal antigen. In even another embodiment, the antigen is a prion antigen. In one aspect, the specific polypeptides that are produced in response to antigen contact (marker proteins) are isolated. These are used to raise antibodies, which are used in subsequent assays involving isolated APCs from patients, whereby expressed polypeptides in the patient isolated APCs are identified, i.e., qualitatively and quantitatively by immunological assays, e.g., ELISA, FACs, RIA and similar techniques.

[00013] In another aspect, the invention provides for determining the proteomic signature of an antigen presenting cell that has been exposed to an antigen. In one embodiment, the proteomic signature is obtained by subjecting the antigen presenting cell to SELDI mass spectroscopy. In another embodiment, the proteomic signature is obtained by subjecting the antigen presenting cell to MALDI-O-TOF and other forms of mass spectroscopy. In yet another aspect the invention provides for proteomic signatures obtained from antigen presenting cells that have been exposed to an antigen. In one embodiment, the antigen presenting cell is a dendritic cell. In yet

another embodiment, the antigen is a cancer antigen. In still another embodiment, the antigen is a viral antigen. In even another embodiment, the antigen is a bacterial antigen. In still another embodiment, the antigen is a fungal antigen. In even another embodiment, the antigen is a prion antigen.

[00014] In one aspect the invention includes a diagnostic method including the steps of: obtaining from a mammal having a disorder, a sample of blood having a subpopulation of antigen presenting cells; substantially isolating the antigen presenting cells from the blood sample; deriving one or more marker polypeptides from the antigen presenting cells, where the marker polypeptide is expressed in the antigen presenting cell in response to antigen contact and where the antigen contacted is associated with or the causative agent of the disorder; obtaining an antibody to the marker polypeptide; and detecting in the antigen presenting cells of a subject, the presence or absence of a polypeptide that binds to the antibody, wherein the presence of the polypeptide confirms the presence of the disorder in the subject. Detection of polypeptides that bind antibodies can be performed using assays such as an ELISA, RIA, FRET, FACs and other immunological detection methodologies. In one embodiment, the antigen presenting cells are dendritic cells. In one embodiment, the disorder is a cancer or cell proliferative disorder. In one embodiment, the disorder is a pathogenic infection. In one embodiment, the disorder is a viral infection. In one embodiment, the disorder is a bacterial infection. In one embodiment, the disorder is a prion infection. In one embodiment, the disorder is a fungal infection.

[00015] In another aspect, the invention includes a method of diagnosing exposure to an antigen comprising the steps of detecting the amount of protein/gene expression present in a sample of mammalian tissue or mammalian body fluids that has not been exposed to the antigen. Then the amount of protein/gene expression present in a sample of mammalian tissue or mammalian body fluids that has been exposed to the antigen is detected. A determination of the difference in the detected amount of protein/gene expression between the exposed and unexposed samples is made. A comparison of the difference to a library of expected protein/gene expression for predetermined antigens is made. Finally, an evaluation is made whether the difference indicates the exposure to a particular antigen. The present invention is particularly useful because it can provide a diagnosis of whether a person

has been exposed to an antigen before the onslaught of any symptoms. The present invention is also directed to a method of diagnosing exposure to an antigen comprising the steps of detecting the patterns of gene expression/proteins present in a sample of mammalian tissue or mammalian body fluids from persons that have been potentially exposed to the antigen, determining the relative amounts of expression of a panel of genes or proteins relative to house keeping genes and proteins expressed in those tissues from the potentially exposed individuals, comparing the relative amount differences to a library of expected gene expression/proteins for predetermined antigens; and evaluating whether the differences indicate that exposure has occurred to a known, catalogued, toxic agent, to a previously unknown antigen, or to a antigen mixed with potentiating agents. Housekeeping genes are genes that tend not to change upon exposure to antigens.

[00016] These aspects and other features will be apparent from the discussion that follows.

DETAILED DESCRIPTION

[00017] The present invention is based on the observed plasticity of antigen presenting cells (APC), and their use for the rapid detection of specific changes in gene and protein expression occurring in human dendritic cells and monocytes in response to exposure to pathogens, tumors, and hazardous agents. 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 the mammalian body. For example, DC cells are found in an immature state in most tissues (CDIa+, CD83 ^Low ), where they recognize and phagocytose pathogens and other antigens (see, Poindexter, et al., (2004) Breast Cancer Res. 6(4):408-415). Platelets, although not cells per se, can also bind and phagocytose infectious microorganisms and serum proteins, and can be considered as a reservoir for the detection of 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 l;99(l l):4021-9). Accordingly, an antigen presenting cell generally refers to those cells and cell fragments that internalize antigens, and possess the capacity to present these antigens to other cells. Exemplary

antigen presenting cells include but are not limited to cells of lymphoid lineage such as T cells, B cells, lymphoid related dendritic cells and natural killer cells, and cells of the myeloid lineage such as myeloid related dendritic cells, macrophages, monocytes, megakaryocytes, platelets, granulocytes and neutrophils. Preferred are highly phagocytotc cells such as macrophages, monocytes and dendritic cells.

[00018] Direct contact with antigens or other pathological agents leads to the maturation of these antigen presenting cells, which is characterized by an increase in antigen presentation, expression of costimulatory molecules, expression of cytokines, and subsequent stimulation of naive T cells in the lymphoid organs, as well as other cell specific markers such as surface CD83 expression in dendritic cells. This maturation process is regulated by numerous corresponding changes in gene expression in these cells, that can be qualitatively and quantitatively measured. The series of gene expression changes that occur are highly specific, and are in specific response to the particular antigen to which the APC is exposed. APC can differentiate between, for example, particular peptides, glycopeptides, glycolipids, and initiate responses that are similar but not identical, when exposed to various antigens. The particular changes in gene and protein expression of the APC in response to antigenic challenge represent very specific measurable biological signatures, which can be used to identify that an APC has experienced an antigen, as well as the nature of the antigen itself, e.g., its chemical composition and source. In certain embodiments, isolation of APC permit the subsequent extraction and isolation of phagocytosed antigens or even whole pathogens from the APC, which can be further characterized by MS or similar tools. For example, DC are known to internalize viral and bacterial pathogens without killing the pathogen (Sundquist et al., 2004, and Jantsch et al., 2003). Accordingly, one object of the present invention includes methods of harvesting parts and/or the entire pathogen or antigen, in addition to obtaining genomic/proteonomic signatures of the pathogen or antigen. Another object of the invention is isolation of the APC polypeptides that are upregulated in response to antigen contact. These polypeptides are highly specific markers for antigen contact, and their expression are indicative that the APC has encountered a particular antigen. By isolating these polypeptides, in whole or in part, antibodies can be raised, which are used in subsequent assays to determine antigen contact.

[00019] For example, see, United States Patent 6,316,197 to Das , et al . ,

Method of diagnosing of exposure to toxic agents by measuring distinct pattern in the levels of expression of specific genes. Exposure of immature DCs to LPS stimulation contributes to their terminal differentiation into CD70(+) DCs (see, 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]). See also, Kumar, A., Kurl, R. N., Kryworuchko, M., Diaz-Mitoma, F., & Sharma, S. 1995. Differential effect of heat shock on RNA metabolism in human Burkitt's lymphoma B-cell lines. Leuk.Res, 19(11): 831-840., which details an association between EBV transformation and enhanced expression of c-myc and poly-A polymerase (PAP) activity. See also, Hwang SL., et al., Indoleamine 2, 3-dioxygenase (IDO) is essential for dendritic cell activation and chemotactic responsiveness to chemokines., Cell Res. 2005 Mar; 15(3): 167-75, describing the upregulation of IDO in response to LPS or TNF-alpha stimulation; and Shi J., et al., Cancer Sci. 2005 Feb;96(2): 127-33., which describes human cord blood monocyte-derived DCs acquiring the ability to kill hematological tumor cells, after activation with lipopolysaccharide (LPS) or gamma- interferon (IFN-gamma), associated with the enhanced TNF-alpha-related apoptosis- inducing ligand (TRAIL) expression in cord blood DC cytoplasm. See also, Smith, A.P., et al., J Virol. 2005 Mar;79(5):2807-13, which details that human herpesvirus-6 (HHV-6), but not the closely related betaherpesvirus HHV-7, dramatically suppressed the secretion of interleukin-12 (IL- 12) p70 by DC, while the production of other cytokines that influence DC maturation, i.e., IL-IO and tumor necrosis factor alpha, was not significantly modified. Komer, et al., described the upregulation of macrophage 103 genes upregulated in response to Bacillus anthracis lethal toxin (LeTx). Similarly, Tucker et al., describes LeTx cleavage of mitogen activated protein kinase kinases (MAPKKs) in a variety of different APC cell types. Expression of genes regulated by MAPKK activity did not change significantly, yet a series of genes under glycogen synthase kinase-3-beta (GSK-3beta) regulation changed expression following LeTx treatment. See also, Green, S. J., Scheller, L. F., Marietta, M. A., Seguin, M. C, Klotz, F. W., Slayter, M., Nelson, B. J., & Nacy, C. A. 1994. Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens. Immunol. Lett, 43(1-2): 87-94, which describes regulation of nitric oxide (NO) production by APC in response to contact with Leishmania major,

tularemia (Francisella tularensis), Mycobacterium bovis (BCG), and Plasmodium berghei. See also, Hemychova, L., Kovarova, H., Macela, A., Kroca, M., Krocova, Z., & Stulik, J. 1997. Early consequences of macrophage-Francisella tularensis interaction under the influence of different genetic background in mice. Immunol. Lett, 57(1-3): 75-81; and Clemens, D. L., Lee, B. Y., & Horwitz, M. A. 2004. Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages. Infect. Immun., 72(6): 3204-3217. See also, Ng, L. C, Forslund, O., Koh, S., Kuoppa, K., & Sjostedt, A. 2003. The response of murine macrophages to infection with Yersinia pestis as revealed by DNA microarray analysis. Adv.Exp.Med Biol, 529: 155-160, which details a total of 22 different genes as up-regulated in response to the Y. pestis infection. These genes include unknown EST's, cytokines, enzyme of cytokine, receptors, ligands, transcriptional factors, inhibitor of transcriptional factor, proteins involved with the cytoskeleton, and 7 genes that encode for factors known to be associated with cell cycling and cell proliferation, with 3 of them playing a role in apoptosis. See also, Saban, M. R., Hellmich, H., Nguyen, N. B., Winston, J., Hammond, T. G., & Saban, R. 2001. Time course of LPS-induced gene expression in a mouse model of genitourinary inflammation. Physiol Genomics, 5(3): 147-160, which details that LPS treatment of APC downregulated the expression transcription factors, protooncogenes, apoptosis-related proteins (cysteine protease), intracellular kinases, and growth factors. Gene upregulation in response to LPS was observed in a cluster including the interleukin-6 (IL-6) receptor, alpha- and beta-nerve growth factor (alpha- and beta-NGF), vascular endothelial growth factor receptor- 1 (VEGF Rl), C-C chemokine receptor, and P-selectin. Another tight cluster of genes with marked expression included the protooncogenes c-Fos, Fos-B, Fra-2, Jun-B, Jun- D, and Egr-1. Almost all interleukin genes were upregulated as early as 1 h after stimulation with LPS. Nuclear factor-kappaB (NF-kappaB) pathway genes collected in a single cluster with a peak expression 4 h after LPS stimulation. In contrast, most of the interleukin receptors and chemokine receptors presented a late peak of expression 24 h after LPS exposure. See also, Mendis, C, Das, R., Hammamieh, R., Royaee, A., Yang, D., Peel, S., & Jett, M. 2005. Transcriptional response signature of human lymphoid cells to staphylococcal enterotoxin B. Genes Immun., 6(2): 84-94.

[00020] Thus these polypeptides, and other APC polypeptides provide for protein markers that are indicative of antigen contact. In one aspect, these polypeptide markers are isolated and used to raise antibodies. The anti-APC marker antibodies are then useful in assays that can be used to detect expression of APC marker polypeptides in cells obtained from patients suspected of antigen exposure. In one embodiment, the anti-APC marker antibodies are used in assays that employ immunological detection methods, such as fluorescent activated cell sorting (FACS), fluorescence resonance emission tomography (FRET), radioimmunoassay (RIA) and enzyme linked immunosorbant assays (ELISA). Other immunological detection assays are know to those of skill in the are and are suitable for the detection methods described herein.

[00021] Accordingly, changes in APC in response to antigenic challenge can be used to assay for persons in presymptomatic (not ill) state, and can be used to monitor the progression of a disease, or the efficacy of a therapeutic regimen in treating the disease. For example see Bernardo, K., Pakulat, N., Fleer, S., Schnaith, A., Utermohlen, O., Krut, O., Muller, S., & Kronke, M. 2004. Subinhibitory concentrations of linezolid reduce Staphylococcus aureus virulence factor expression. Antimicrob. Agents Chemother., 48(2): 546-444, which describes the influence of the antibiotic linezolid on the secretion of exotoxins by Staphylococcus aureus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot analysis. Similarly, changes in APC in response to antigenic challenge can be used to assay for persons who have been exposed to biological agent(s)- and can be used in early diagnosis of the high risk exposed individual; as well as for monitoring persons who are in the early stages of developing symptoms. For example, changes in APC in response to antigenic challenge from a viral or bacterial pathogen can provide for rapid identification of these pathogens, and may predate the eventual pathogen appearance in plasma by many hours or days. In a related application, the invention can be used to monitor the effectiveness of a vaccination, by assaying for DC interaction with one or more components of the vaccine. Similarly, changes in APC in response to antigenic challenge from tumors permit the detection of tumors before an individual becomes symptomatic, thereby permitting early aggressive treatment. Also, changes in APC in response to exposure to

industrial chemicals, or biowarfare agents may provide for identification of the unknown etiological agent to which an individual may be exposed.

[00022] APCs serve as the body's natural immune biosensor. These cell types circulate through all tissues of the body and are responsible for surveying most if not all tissues of the body by sampling the microenvironment. In doing so, they seek out areas of tissue that have a danger signal, i.e., increased mitotic activity, or viral/bacterial infections (Crawford et al., 2003). Once this signal is detected, APCs, initiate the early transcriptional changes, which lead to cell surface antigen expression and inflammatory mediator release (Crawford et al., 2003). These cellular modifications are required for recruitment of other inflammatory cells to the site of involvement and improved immune cell-to-cell contact. Antigen presenting cells such as DCs possess pattern recognition receptors, which allow them to bind to and discriminate between various pathogens (Chaussabel et al., 2003). Other receptors include Toll-like receptors, ICAM's such as ICAM-I, DCSIGN, and others.

[00023] As described above, APCs generate unique gene signatures in response to exposure to various pathogens. Studies of discordant gene expression in DC and macrophages infected with bacteria, Candida, influenza, or different parasites using oligonucleotide arrays have suggested that of the approximately 6800 genes samples, about 1300 genes demonstrate significant modulation in expression patterns after exposure to antigens (see, Huang et al, The plasticity of dendritic cell responses to pathogens and their components, Science, 294: 2001). For example, DC express C- type lectins as pathogen recognition receptors, for example, the DC-specific ICAM-3 grabbing nonintegrin (SIGN)/CD209, which has been identified as the HIV-I receptor on DC, as well as for surface glycans for Mycobacterium tuberculosis, Helicobacter pylori, Leishmania mexicana, Schistosoma mansoni, and other pathogens (see, Appelmelk et al. (2003), J. Immunol., 170:(4): 1635-9). See also Hofer et al., (2001) Immunol. Rev., Jun:181:5-19, and Pulendran et al., (2001), J. Immunol. Nov.: 167(9):5067-76. These receptors and the cellular pathways they interact with, provide unique markers for monitoring DC activation and response. More particularly, changes in DC response are measurable and provide pathogen specific signatures evidencing DC interaction with particular disease agents. See, Machein, U. & Conca, W. 1997. Expression of several matrix metalloproteinase genes in human

monocytic cells. Adv.Exp.Med Biol, 421 : 247-251., detailing several MMP genes are transcriptionally active in the cells tested after exposure to a variety of stimuli such as phorbol ester, lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB).

[00024] Hazardous environmental agents are also detectable by the methods described herein, as they either can provoke an APC specific immune cell response themselves, or will destroy cells and tissues causing an increase in inflammation, extravasation, and activation of APCs in response to cytokines and various cellular factors. These properties of human APCs make them suitable for the rapid detection of exposure to any pathogenic substance, for example an infectious pathogen, tumor, toxin or toxic industrial chemical (TIC), or weapon of mass destruction (WMD).

[00025] The APCs described, preferably monocytes, and most preferably DC are useful to detect changes in the physiology of a subject, in response particularly to diseases, such as infectious diseases and cancers. As used herein, the term antigen is broadly used to refer to any composition that is generally foreign to a healthy mammal, or is native to the mammal but is mutated, aberrant or found in increased concentrations in the mammal having a pathological condition. An antigen thus includes whole pathogens such as bacteria, viruses, fungi, protozoa, as well as one or more components of a pathogen, for example a bacterial antigen includes lipopolysaccharide (LPS), a viral antigen includes a viral coat protein such as gpl20 of HIV or hemagglutinin of influenza, and a fungal antigen includes the cell- wall derived protein mannin. Prions are also antigenic, displaying specific peptide sequences associated with disease states. Antigens also include proteins and peptides associated with tumors, such as carcinoembryonic antigen (CEA) and aberrantly glycosylated mucin (MUC) as well as numerous other tumor specific antigens and proteins such as bcl-2, survivin, hepsin and the like. Accordingly, the common characteristic of an antigen, or antigenic agent, is the effect it has on an APC in that it causes specific biochemical changes in the APC such as the upregulation of antigen presentation proteins and co-receptors, as well as causing maturation and proliferation of APCs, tissue migration, and other properties that are indicative of exposure to an antigen. A detectable increase in APC is one where for example a two-fold or greater increase in the number of APCs are induced to develop or activate or mature in the mammal exposed to the antigen relative to those levels of APCs in the non-exposed

or healthy mammal. Assay techniques for determining DC and other blood cells are well known in the art, for example but not limited to FACS using mature DC cell markers CD2+ and CD83+, or immature marker CDIa+.

[00026] In one aspect, the invention provides immune cell based methods for monitoring a patient's response to a disease state. Determining the pathogen or tumor-specific genomic and proteomic expression patters, or signatures, provides an improved method of on-going monitoring of the patient's immune response to the disease state. This information is used in conjunction with other relevant medical information such as decrease in tumor mass or tumor burden for a cancer patient, or a decrease in viral load for an HIV infected patient, or the clearance of mycobacteria in a tuberculosis patient, to allow monitoring of therapeutic efficacy, for example, in response to chemotherapy, anti-viral therapy, or administration of antibiotics.

[00027] APCs thus provide a useful diagnostic tool for identifying antigens and for monitoring the health of individuals, based upon changes in their cellular metabolism. Measurable changes occur in expression of numerous genes, proteins and secretory factors such as cytokines, and the antigens can also be detected in the cytoplasm of the APC (such as in the cytoplasm of platelets). As such, in one aspect the present invention provides for arrays of APC gene signatures, preferably monocyte or DC signatures. The array includes oligonucleotides, oligoribonucleotides or polypeptides of a plurality of APC marker genes and proteins, i.e., gene or protein products differentially expressed in antigen presenting cells. More preferably, the array includes from about 500-1000 specific markers at individual addresses in a matrix. Even more preferably, the array includes about 5,000, about 10,000, about 20,000 or greater genes or gene products, represented on the array. Most preferably, the array is a genome wide array, for example 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 controls or for quantitative analysis of gene expression. The manufacture and use of such arrays are described in United States Patent 6,741,344, 6,733,977, and 6,733,964. A method and apparatus for selectively applying a material onto a substrate for the synthesis of an array of, for example, oligonucleotides at selected regions or addresses on the substrate is further described by U.S. Patent number

6,667,394. The gene arrays produced are representative of the host reaction to the pathogen in great detail (typically 52,000 genes or more) and are not dependent the identification of one or a few genes (intrinsically biased), as is the case for identification by, e.g., Q-PCR. Proteomic data can be developed by a variety of techniques, for example but not limited to using surface plasmon resonance, or mass spectroscopy (MALDI or SELDI, etc). The combination of information obtained using genomic and proteomic approaches, in the format of a high throughput screen such as a DC gene array provides exceptionally specific diagnostic data, and thus a powerful tool for antigen identification or patient monitoring.

[00028] Numerous types of arrays are created, to develop APC based diagnostic arrays for a variety of purposes, but generally to obtain data sets for how APCs, particularly DCs, react upon exposure to different antigens. The use of a particular array depends on its chemical composition, and will vary depending on weather the array has nucleic acids, peptides, both, or other chemical moieties such as lectins etc. By way of general illustration, arrays such as Affymetrix's GeneChip® use biotin labeled cRNA prepared from cell extracts. About 5 micrograms total RNA are an appropriate starting material. The cRNA produced from the RNA sample is exposed to the array, allowed to hybridize to the appropriate target. The array is then washed and stained, e.g., with streptavidin phycoerythrin, then visualized using Affymetrix's GeneChip® Scanner 3000 or an Agilent GeneArray® Scanner. This technique as well as known immunological methods and other common methods of using proteomic and genomic arrays will be generally understood to those skilled in the art.

[00029] The arrays provide for the detection and identification of pathogens and pathogenic agents, as well as the detection and identification of transformed cells and tissues, using samples derived from subjects. Information about the disease state of a patient, that is, a patient data set, is obtained using one or more of the APC arrays described, by first obtaining a sample of blood from a subject, and then isolating the DCs from that blood sample. The DC signature from the patient is compared to one or more control DC signatures, for example, using the hybridization arrays described. The control DC signatures on the array minimally represent both the normal or healthy DC signatures and the abnormal or pathogenic 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, e.g., cancer stages. The arrays and the data sets obtained there from are useful, for example, for discovering or diagnosing the existence of a genetic disease or chromosomal abnormality, or to provide information relating to identity, heredity or compatibility, diagnosing a predisposition to a disease or condition, diagnosing infection by a pathogenic organism, discovering or diagnosing neoplastic transformation of a cell or tissue, determining exposure to and identification of biowarfare or chemical warfare samples, or toxic industrial chemicals.

[00030] In one aspect, APC arrays are developed that are designed to identify the presence or absence of particular pathogens as well as their immunological consequences during the progression of the disease state they are associated with. For example, arrays are created that provide for the detection and monitoring of a viral infection such as HIV. The arrays include consensus APC signatures from immature or naive APCs, from APCs obtained from an HIV exposed but asymptomatic person, APCs obtained from the exposed and early symptomatic person and from APCs in the later stage symptomatic person. Similar viral arrays are developed, for example ones 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 in gene therapy are also preferred, e.g., those directed to vaccinia or poxviruses, and more particularly, those specific to the transformed vector, which should produce a different DC signature than the wild type vector. In yet another aspect, the arrays include human APC, particularly DC and macrophage cells that are exposed to pathogens on CDC priority list. These types of arrays will facilitate rapid emergency diagnosis, etiologic studies, response and treatment of exposed or potentially exposed individuals. Arrays specific to homeland defense or military uses are also provided herein, as DC arrays specific to biological warfare pathogens provide for rapid detection and response to terrorist or enemy bioweapons attacks. Such arrays include smallpox arrays, bacillus anthracis arrays, and other WMD pathogens. For example, arrays of human APC, particularly DC and macrophage cells that are exposed to toxic agents facilitates emergency diagnosis, response and treatment of exposed or potentially exposed individuals. In another

embodiment, the array and patient data set obtained there from facilitate forensic or toxicology studies of an exposed individual.

[00031] In another aspect, the arrays are obtained from human APC, particularly DC and macrophage cells in patients having different tumors, including different stages of tumor growth. APC arrays are designed to identify the presence or absence of particular tumor antigenic markers, and the immunological consequence of the tumor on the patient during the progression of the patient's cancer. This type of array facilitates rapid diagnosis, tumor identification, and appropriate treatment of afflicted individuals. The following cancer types each result in specific APC responses, and are amenable to detection using the techniques described: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Acute Myeloid Leukemia, Childhood; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Cancers; AIDS- Related Lymphoma; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas/Carcinoids, Childhood; Carcinoid Tumor, Childhood; Carcinoid Tumor,Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma/Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-CeIl Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor;

Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer,Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Childhood Brain Stem; Glioma, Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin'sLymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non- Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-CeIl; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's, Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non- Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During

Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood; Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non- Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma)/Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-CeIl Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma and Thymic Carcinoma Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Carcinoma of, Adult; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor.

[00032] Arrays are created that provide for the detection and monitoring of various cancers such as breast cancer, colon cancer, ovarian cancer, uterine cancer, prostate cancer, glioma, melanoma, small and large cell carcinoma, leukemia, and

other neoplastic and precancerous disease states. Markers such as aberrantly glycosylated MUC-I, or expression of CEA or hepsin are examples of common tumor markers known to be associated with most of the above tumors. Comprehensive listings including tumor-specific markers are known in the medical literature. Exemplary arrays include consensus APC signatures from immature or naϊve APCs, and from APCs obtained from persons having stage 0, 1, 2, 3 or 4 graded tumors. Histological profiles and other medical data may be used in connection with the APC arrays to provide additional information about the disease state. The plasticity and specificity of response of APCs to cancers allows very specific identification of the cancer type, and the staging of the disease. As such, they also permit a medical professional to monitor the course of a therapeutic regimen, by monitoring changes in APC signatures during, for example, a chemotherapy regimen. For example, a patient with pancreatic cancer is provided with gemcitabine, and before and during the course of gemcitabine therapy DCs are extracted to the patient and used with a pancreatic cancer DC array. The array indicates the patient had grade 3 pancreatic cancer at the outset of the treatment, and indicates that one moth of gemcitabine treatment has caused the cancer to revert to a grade 2 stage, thereby indicating continued gemcitabine therapy for the patient.

[00033] In even another embodiment, arrays of APCs from individuals having a genetic disorder are created. Representative genetic disorders include for example, a disease state resulting from the presence of a gene, the expression product of the gene being a bioactive molecule that causes or contributes to the disease state, or the absence of a gene where the expression product of the gene in a healthy individual is a bioactive molecule that ameliorates or prevents the disease state. An example of the former is cystic fibrosis, wherein 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 the lack of an enzyme permitting the metabolism of phenylalanine. Examples of genetic disorders appropriate for screening with the present assays and methods include, for example multiple sclerosis, endocrine disorders, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Lupus, Angelman syndrome, Charcot-Marie-Tooth disease, Epilepsy, Essential tremor, Fragile X syndrome, Friedreich's Ataxia, Huntington disease, Niemann-Pick Disease, Parkinson's Disease, Prader-Willi syndrome, Rett syndrome, spinocerebellar atrophy, Williams syndrome, Ellis-van Creveld syndrome,

Marfan Syndrome, Myotonic dystrophy, leukodystrophy, Atherosclerosis, Best disease, Gaucher disease, glucose galactose malabsorption, Gyrate atrophy, Juvenile onset diabetes, Obesity, Paroxysmal nocturnal hemoglobinuria, Phenylketonuria, Refsum disease, and Tangier disease. Such arrays are useful in detecting a genetic disorder in a patient, and monitoring the patient having the genetic disorder during therapy. Similarly, the present assays provide for monitoring the course of gene therapy treatments, by monitoring the immunological state of the patient so treated, particularly for the appearance of the healthy gene product or for adverse reactions to the gene therapy vector.

[00034] While the above discussion has focused on using APCs in diagnostics to determine biological changes in a sample, in another embodiment, direct analysis of the fluids of a subject, such as blood, sputum, urine, saliva, mucus, cerebrospinal fluid, lymphatic fluid and the like can be subjected to assay. These samples are analyzed using various medical techniques, e.g., spinal tap to look for infection in cerebrospinal fluid, or laboratory techniques such as proteomic tools e.g., mass spectroscopy, and are generally known and will also be described below. Thus, the assays of the present invention may involve the screening of APCs for changes in conjunction with direct analysis of the bodily fluids of a subject provides an even more sophisticated detection and monitoring method.

[00035] APCs thus provide a highly specific and rapid means for monitoring biological changes in an organism, based on specific genomic and proteomic signatures that are typified by the DC in a particular state. The above discussion has centered on using APCs in assays that employ such common techniques as hybridization or immunological reactivity. Other proteomics tools are appropriate in determining changes in APC states. One particularly preferred method of obtaining a DC proteomic signature involves obtaining the mass spectra of the APC sample.

[00036] During the last decade, mass spectrometry (MS) has become an important analytical tool in the analysis of biological macromolecules. Mass spectrometry provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them "fly" by volatilization. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). To perform MS, the samples under study

are subjected to Energy Desorption/Ionisation (EDI) from a surface by input of energy. Typically EDIs are thermal desorption/ionisation (TDI), plasma desorption/ionisation (PDI) and various kinds of irradiation desorption/ionisation (IDI) such as by fast atom bombardment (FAB), electron impact, etc. Where a laser is used to ionize the sample, the process is called laser desorption/ionisation (LDI), such as matrix assisted laser desorption/ionisation (MALDI). Desorption may be assisted by presenting the MS analyte together with various helper substances or functional groups on the ionization surface, preferably such as surface-enhanced laser desorption/ionisation (SELDI).

[00037] For molecules of low molecular weight, mass spectrometry has long been part of the routine physical-organic repertoire for analysis and characterization of organic molecules by the determination of the mass of the parent molecular ion. Introduction of the so-called "soft ionization" methods, namely MALDI and ElectroSpray Ionization (ESI), permitted intact ionization, detection and exact mass determination of large molecules, i.e. well exceeding 300 kDa in mass, such as peptides and proteins (see, Fenn, J. B., et al., (1989) Science 246, 64-71; Karas M. & Hillenkamp F. (1988) Anal. Chem. 60, 2299-3001). In addition, by arranging collisions of the ionized parent molecule with other particles (e.g., argon atoms), the ionized parent molecule is fragmented, forming secondary ions by collision induced dissociation (CID). The fragmentation pattern/pathway very often allows the derivation of more detailed information, for example structural information about the molecule.

[00038] MALDI-MS and ESI-MS have been used to analyze nucleic acids as well as proteins (see, Nordhoff E., et al., (1997) Mass Spectrom. Rev. 15: 67-138). However, since nucleic acids are very polar biomolecules, that are difficult to volatize, there has been an upper mass limit for clear and accurate resolution. ESI would seem to be superior to MALDI for the intact desorption of large nucleic acids even in the MDa mass range (Fuerstenau S. D. & Benner W. H. (1995). Rapid Commun. Mass Spectrom. 9, 1528-38; Chen R., Cheng X., Mitchell et al., (1995). Anal. Chem. 67, 1159-1163).

[00039] A few reports on the MALDI-MS of large DNA molecules with lasers emitting in the ultraviolet (UV) have been reported (Ross P. L. & 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 Y-H-, et al., (1995) Anal. Chem. 67: 3482-3490 and Siegert C. W., et al., (1997) Anal. Biochem. 243, 55-65. However, based on these reports it is clear that analysis of nucleic acids exceeding 30 kDa in mass by UV-MALDI-MS gets increasingly difficult with a current upper mass limit of about 90 kDa (Ross P. L. & P. Belgrader (1997) Anal. Chem. 69: 3966-3972). The inferior quality of the DNA UV-MALDI- spectra has been attributed to a combination of ion fragmentation and multiple salt formation of the phosphate backbone. Since RNA is considerably more stable than DNA under UV-MALDI conditions, the accessible mass range for RNA is up to about 150 kDa (Kirpekar F., et al., (1994). Nucleic Acids Res. 22, 3866-3870).

[00040] The analysis of nucleic acids by IR-MALDI with solid matrices

(mostly succinic acid and, 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). The 1992 Nordhoff et al., paper reports that a 20- mer of DNA and an 80-mer of RNA were about the uppermost limit for resolution. The 1993 Nordhoff et al. paper, however, provides a distinct spectra for a 26-mer of DNA and a 104-mer of tRNA. The 1995 Nordhoff et al., paper shows a substantially better spectra for the analysis of a 40-mer by UV-MALDI with the solid matrix, 3- hydroxy picolinic acid, than by IR-MALDI with succinic acid (See FIGS. l(d) and l(e)). In fact the 1995 paper reports that IR-MALDI resulted in a substantial degree of prompt fragmentation.

[00041] In a Time-Of-Flight (TOF) mass spectrometer, the mass-to-charge ratio m/z of ions can be determined from their time of flight. Although it is always the mass-to-charge ratio m/z which is measured in mass spectrometry, with m being the mass and z being the number of elemental charges carried by the ion, in the following, for the sake of simplicity, only the mass m and its determination will be referred to. Since many types of ionization, such as MALDI, predominantly supply only single- charged ions (z=l), the difference ceases to exist in practice for these types of ionization. In a time-of-flight mass spectrometer (TOF-MS) which is equipped with an ion selector and a velocity-focusing reflector, it is possible to measure the

daughter-ion or fragment-ion spectra of parent ions which are selected by the ion selector on the basis of their time of flight. The decay of parent ions into daughter or fragment ions can be induced by introducing excess energy during ionization (so- called PSD "Post Source Decay" spectra) or by applying other methods such as collisionally induced fragmentation. The parent ions and the daughter ions resulting from their decay enter the reflector simultaneously with the same average velocity but with different mass-proportional energies, such that they will be dispersed according to their mass within the reflector by their different energies.

[00042] Thus, mass spectroscopy, as well as other tools that permit detection of e.g., the infrared and ultraviolet absorption spectra, nuclear magnetic resonance spectra, as well as analytical profiles such as biomolecular interaction analysis (e.g., ELISA or surface plasmon resonance (SPR) profiles, see, Nedlekov et al., (2003) Appl. Env. Microbiol.) and other techniques to measure the physical properties of a sample, also provide methods for analyzing the samples. The information obtainable from methods using APCs described above, in connection with traditional laboratory methods, provides an integrated approach leading to the ability to resolve different properties of each sample under study. For example, where MS profiles for two samples display highly similar patterns, a second analysis such as an IR spectra, NMR spectra or SPR is used to provide additional comparative signatures and information. The result is an analytical signature profile, specific for each sample or sample under analysis, that provides for independent identification of the sample, (alone or in a mixture), and which can also provide, in certain embodiments, quantitative information about the sample such as concentration, as well as qualitative information, such as identification of other agents or materials in the sample mixture.

[00043] A preferred method utilizes mass spectroscopy to obtain proteomic signatures of APCs in healthy states and in response to challenge with antigens. Mass spectroscopy can also be used directly on mixtures suspected of containing the antigens or other contaminants. Most preferred analytical methods for obtaining these signatures includes SELDI, such as Ciphergen's ProteinChip® System Series 4000, or MALDI O-TOF, based on an orthogonal platform coupling the MALDI to the MS, such as Perkin Elmer's prOTOF™ 2000 MALDI O-TOF Mass Spectrometer.

[00044] In one aspect, proteomic signatures of APC, preferably DC, are obtained after challenge from toxins and organisms on the National Institute for Allergy and Infectious Diseases Biodefense Priority Pathogens List. The DC are cultured with the antigens or fragments thereof, as is described below. Proteomic signatures are obtained. These relevant antigens include Bacillus anthracis (anthrax), Clostridium botulinum, Yersinia pestis, Variola major (smallpox) and other pox viruses, Francisella tularensis (tularemia), and those causing Viral hemorrhagic fevers, Arenaviruses, such as LCM, Junin virus, Machupo virus, Guanarito virus, and those causing Lassa Fever, Bunyaviruses and Hantaviruses such as those causing Rift Valley Fever, Caliciviruses, Hepatitis A, B and C), viral encephalitides such as West Nile Virus. LaCrosse, California encephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus, Kyasanur Forest Virus, Tickborne hemorrhagic fever viruses, Crimean-Congo Hemorrhagic fever virus, Tickborne encephalitis viruses, Yellow fever, Multi-drug resistant TB, Influenza, Other Rickettsias and Rabies, Flaviruses, Dengue, Filoviruses, Ebola, Marburg Burkholderia pseudomallei, Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), Ricin toxin (from Ricinus communis), Epsilon toxin of Clostridium perfringens, Staphylococcus enterotoxin B, Typhus fever (Rickettsia prowazekii), Food and Waterborne Pathogens, Bacteria such as Diarrheagenic E.coli, Pathogenic Vibrios, Shigella species, Salmonella, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica), and Protozoa such as Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma, Microsporidia. These biosamples are amenable to detection and identification, based on such criteria as lipoprotein content, glycoprotein content, membrane composition, the presence and absence of viral envelopes, expression of particular proteins such as virulence factors, and other biochemical profiles. See, for example, Dell A, Morris HR, Glycoprotein structure determination by mass spectrometry. Science. 2001; 291(5512):2351-6. See also, Rudd P.M., et al., Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc Natl Acad Sci U S A. 1999, 96(23): 13044-9. See also, 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 obtained from samples of cells, fluids and tissues of a subject exposed to (or suspected of exposure to) one or more toxins and organisms on the National Institute for Allergy and

Infectious Diseases Biodefense Priority Pathogens List. The signatures of DC and fluids or tissues are compared to reference signatures to confirm exposure and to aid in monitoring treatment. For example, a blood sample is obtained from a subject suspected of having been exposed to smallpox. The sample is split into two aliquots; the DC recovered from one, and the plasma purified from the other. Both samples are subjected to mass spectroscopy. The DC signature is compared to reference signatures that provide positive and negative controls for exposed and naive DC, and the plasma is assayed for the presence of variola virus. The signatures can confirm infection, before the patient becomes viremic or symptomatic, thus facilitating their quarantine.

[00045] The SELDI or MALDI O-TOF mass spectrometer signatures may profile the nucleic acids, proteins, carbohydrates and lipids of a microbial sample, but can preferably profile and obtain a signature for the whole pathogen. The signatures distinguish between microbial species, and varieties within the species, e.g., E. coli 0157, stages of microbial growth, e.g., sporulative, vegetative, or in active growth, and relative age, as well as other characteristics such as pathogenicity, for example the pyrogenic exotoxin A production in group A streptococci, the cholera toxin in Vibrio cholerae, Shiga toxin-producing Escherichia coli (STEC), or enterotoxin production in enterohemorrhagic (EHEC) strains of E. coli. For example, Lai et al., discuss that sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analyzed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded by Lai et al., that FTIR is a reliable method for investigating Carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification. See, Lai, S., R. Goodacre, et al. (2004). "Whole-organism fingerprinting of the genus Carnobacterium

using Fourier transform infrared spectroscopy (FT-IR)." Syst Appl Microbiol 27(2): 186-91. Similarly, Lee et al discuss a bacterial analysis method coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix- assisted laser desorption/ionization time-of-flight mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI- MS. Each fraction, collected over different time intervals, corresponded to different sizes and different growth stages of bacteria. See, Lee, H., S. K. Williams, et al. (2003). "Analysis of whole bacterial cells by flow field- flow fractionation and matrix- assisted laser desorption/ionization time-of-flight mass spectrometry." Anal Chem 75(11): 2746-52. Likewise, Lefmann et al., discuss MALDI-TOF MS after base- specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA), used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS- based comparative sequence analysis, mycobacterial 16S rDNA signature sequences (approximately 500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase- mediated transcription of forward strands in the presence of 5 -methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase Tl, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. Lefmann et al., concluded that analysis of 16S rDNA amplicons by MS after base- specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). See, Lefmann, M., et al., Novel mass spectrometry-based

tool for genotypic identification of mycobacteria. J Clin Microbiol 42(1): 339-46 (2004). Thus, one object of the present invention include obtaining proteomic, genomic, lipid, carbohydrate, and whole organism signatures for bacterial pathogens. Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry, alone or in conjunction with other assays. Microbial identification is not limited to bacteria, and the analytic signatures of other pathogenic organisms thus include those of fungi, viruses, prions, and other infectious agents and pathogens.

[00046] The proteomic signatures derived from APC and those obtained by direct assessment of the pathogens from the fluids of a patient are used in the diagnosis of disease as described, but are particularly useful for monitoring the course of therapy, e.g., in response to antimicrobial compounds such as terbinafine, fluconizole, lamivudine, ciprofloxacin, vancomycin, penicillin, methicillin and other antibiotics. Signatures of tissues, fluids and cells of a subject therapeutically treated with antimicrobial compounds can also be analyzed for toxicity during such therapy. Detection of viral samples is described in, for example, Hong et al., which assayed for mutations in hepatitis B virus (HBV) permitting lamivudine resistance, that arise during prolonged treatment with that drug. Therapy with lamivudine frequently causes selection for HBV virions having amino acid substitutions in the YMDD motif of HBV DNA polymerase. MALDI-TOF MS genotyping detects HBV variants in a sensitive and specific manner. The assay in Hong et al., is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif. The MALDI-TOF MS-based genotyping assay described therein is sufficiently sensitive to detect as few as 100 copies of HBV genome per milliliter of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS- based assay correctly identified known viral variants and additional viral quasi- species not detected by previous methods, as well as their relative abundance. Hong et al., concluded the sensitivity, accuracy and amenability to high- throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy. See, Hong et al., Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments, J Hepatol. (2004). Thus, one object of the present invention includes the proteomic,

genomic, lipid, carbohydrate, and whole organism signatures for viral pathogens, and analytic signatures of DC and other tissues, fluids and cells of a subject having a viral infection. Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry, alone or in conjunction with other assays.

[00047] Bonetto et al., discusses the elucidation of the structure and biological properties of the prion protein scrapie (PrP(Sc)) as fundamental to an understanding of the mechanism of conformational transition of cellular (PrP(C)) into disease- specific isoforms and the pathogenesis of prion diseases. They observed that a construct of 106 amino acids (termed PrP 106 or miniprion), derived from mouse PrP was highly toxic to primary neuronal cultures, and induced a remarkable increase in membrane microviscosity. See, Bonetto V., et al., Synthetic miniprion PrP106, J Biol Chem. 277(35):31327-34 (2002). Accordingly, in still another aspect, the invention includes signatures of prion samples, and signatures of APC, and tissues, fluids and cells of a subject having a prion infection. Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry, alone or in conjunction with other assays.

[00048] It is yet another object of the invention to obtain the signatures, e.g.,

SELDI or MALDI O-TOF MS and other analytic signatures, from healthy and from diseased subjects, i.e., APC, fluids and tissues, for example in diseases characterized by various stages of physical degeneration, such as, cardiac muscle, kidney, or neural tissues, in various stages of infection, such as viral or bacterial, or in various stages of transformation, malignancy or tumorogenicity. In particular, cancers and premalignant tissues all undergo significant biochemical changes relative to nondiseased cells and tissues, that can be readily detected by spectral and other types of analytical methods. One example of this is the change in glycosylation patterns seen in the tumor associated antigen MUC- 1 in many different cancers types, or the differential expression of chorioembrionic antigen (CEA), or tumor suppressor genes such as retinoblastoma (RB), p53, and cyclin dependent kinases cdk's. Numerous markers for cellular transformation and cancer are known in the medical literature, and all of these can be disease signatures for the purpose of the present invention. Likewise, these tissues exhibit changes to their metabolic states in response to treatment with chemotherapeutic samples and radiation. These changes are molecular signatures of a response to treatment, and are thus useful for the purposes described

herein. Thus, the present methods can be used, for example, to identify and stage a particular tumor type, and monitor changes to the tumor over the course of therapy, such as chemotherapy or radiation. The methods described may also be used to monitor changes to healthy organs and tissues during such chemotherapy or radiation regimens, for example, to assess the systemic toxicity of the therapy for making adjustments to the course of treatment. In one embodiment, a toxicology profile for a chemotherapy regimen is provided. This profile comprises tissue specific molecular analytical signatures of a plurality mammalian organs and tissues in an untreated state, i.e., without exposure to a chemotherapy drug, as well as in response to a plurality of dosages of the drug. The profile can include a time dimension, i.e., dose response signatures of the tissues over a period of time. The present invention thus includes analytical signatures useful in the detection and treatment of disease. For example, Chaurand et al., determined that analysis of thin tissue sections of organs results in over 500 individual protein signals in the mass range of 2 to 70 kDa that directly correlate with the protein composition within a specific region of the tissue sample. Such profiling, including imaging MS, has been applied to multiple diseased tissues, including human gliomas and non-small cell lung cancer. Interrogation of the resulting complex MS data sets has resulted in identification of both 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. Likewise Ahmed et al., discuss differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In Ahmed, a total of 24 serum proteins were differentially expressed in grade 1, 31 in grade 2, and 25 in grade 3 ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry as isoforms of haptoglobin- 1 precursor (HAPl), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting and immunohistochemical localization using monoclonal antibodies against a haptoglobin epitope contained within HAPl. 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., discuss detection of serum tumor biomarkers at an earlier stage in order to improve

the overall survival of cancer patients. Utilizing MALDI-TOF-Mass Spectrometry (MS) based protein identification techniques, a SCLC specific overexpressed protein was identified to be haptoglobin alpha-subunit, with its serum level correlating with the disease stage. The mean level of alpha-haptoglobin was increased in SCLC serum as compared to the normal controls. Serum HGF was also studied as potential tumor biomarker and was found to correlate with the disease status. See, Bharti A, et al., Haptoglobin alpha-subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer. Anticancer Res. 2004 Mar- Apr;24(2C): 1031 -8. Several other tumor types are amenable to detection using the present methods. Iwadate et al., discuss the detection and response to chemotherapeutic treatment of gliomas. The biological features of gliomas, which are characterized by highly heterogeneous biological aggressiveness even in the same histological category, are precisely described by global gene expression data at the protein level. Iwadate et al., investigated whether proteome analysis based on two- dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time- of-flight mass spectrometry could identify differences in protein expression between high- and low-grade glioma tissues. Proteome profiling patterns were compared in 85 tissue samples: 52 glioblastoma multiform, 13 anaplastic astrocytomas, 10 astrocytomas, and 10 normal brain tissues. Iwadate et al., could completely distinguish the normal brain tissues from glioma tissues by cluster analysis based on the proteome profiling patterns. Proteome-based clustering significantly correlated with the patient survival, and they could identify a biologically distinct subset of astrocytomas with aggressive nature. Iwadate et al., found that discriminant analysis extracted a set of 37 proteins differentially expressed based on histological grading. Among them, many of the proteins that were increased in high-grade gliomas were categorized as signal transduction proteins, including small G-proteins. Immunohistochemical analysis confirmed the expression of identified proteins in glioma tissues. See, Iwadate Y, et al., Molecular classification and survival prediction in human gliomas based on proteome analysis. Cancer Res. 2004 Apr l;64(7):2496-501. Friedman et al., discuss two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS), used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. Friedman et al., investigated over 1500 protein spot- features in each paired normal/tumor comparison, and using DIGE technology with the mixed-

sample internal standard, and made statistically significant quantitative comparisons of each protein abundance change across multiple samples simultaneously. Matrix- assisted laser desorption/ionization-time of flight and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. See, Friedman DB, et al., Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics. 2004 Mar;4(3):793-811. Hamler et al., discusses a two- dimensional liquid-phase separation scheme coupled with mass spectrometry (MS) for proteomic analysis of cell lysates from normal and malignant breast epithelial cell lines. Liquid-phase separations consist of isoelectric focusing as the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS- RP-HPLC) as the second dimension. Protein quantitation and mass measurement 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 mass maps that allowed visualization of protein quantitation differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins observed from mass mapping experiments over the limited pH range, 40 (36%) were positively identified by peptide mass fingerprinting and assigned to bands in the mass maps. Of these 40 proteins, 22 were more highly expressed in one or more of the malignant cell lines. These proteins represent potential breast cancer biomarkers that could aid in diagnosis, therapy, or drug development. See, 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. Veenstra et al., discusses serum protein fingerprinting. Many proteomic studies have focused on the identification and subsequent comparative analysis of the thousands of proteins that populate 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. Accordingly, these proteomic, carbohydrate, nucleic acid, and lipid spectroscopic profiles or patterns provide for signatures of numerous tissues in both healthy and disease states, and from a diagnostic perspective indicate the presence of

disease and can be used to monitor changes in the organism having the disease. In one embodiment, serum or lymphatic samples are used for obtaining such signatures. In another embodiment, DC are used. In other embodiments, APC provide the analytical signatures. In yet other embodiments, blood cells, muscle tissues, nervous tissues, epithelial tissues and connective tissues are assessed.

[00049] Another object of the present invention includes determining the chemical signatures of toxic industrial chemicals, and the consequential proteomic, genomic, lipid, and carbohydrate signatures of APC, tissues, fluids and cells of a subject that has been, or is suspected of being exposed to toxic industrial chemicals (TIC). Preferably these are obtained using SELDI or MALDI O-TOF mass spectrometry, alone or in conjunction with other analytical methods. The resultant signatures are stored in a database, and made available for diagnostic and therapeutic applications. A toxic industrial chemical is generally understood as a material that has a toxicity (LC50 by inhalation) of less than 100,000 Mg per min/M3 and an appreciable (undefined) vapor pressure at 2O ⁰ C. The term TIC as used herein, includes Toxic Industrial Materials (TIM), generally regarded as any substance that in a given quantity produces a toxic effect in exposed personnel through inhalation, ingestion, or absorption. Examples of TICs and TIMs include fuels, oil, 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. Many TICs and TIMs are known in industry, and the above referenced agents are not intended to be comprehensive or limiting.

[00050] In another aspect, the invention provide for signatures, e.g., chemical, proteomic, genomic, lipid, carbohydrate, and whole organism signatures of agents of significance to national defense, such as biowarfare and chemical warfare agents (also known as WMD), and the proteomic, genomic, lipid, and carbohydrate signatures of APC, tissues, fluids and cells of a subject that has been, or is suspected of being exposed to such agents. Preferably these signatures are obtained with SELDI, MALDI O-TOF MS and other analytic methods. In particular, since spectral analysis provides a rapid and accurate detection means, it is possible to employ the present

invention as part of a rapid or first response program, for field identification of biowarfare and chemical warfare agents in the samples.

[00051] The first step in the analytical process includes obtaining a sample of the agent (TIC, WMD) bacteria, virus, prion, cell, APC, fluid or tissue under study. The sample may be processed prior to examination, i.e., dissolved in water or a solvent, or used intact. Simple analytical methods may be used to gain rudimentary information about the sample. Collection of a mass spectrum and analysis thereof follows. The sample is applied to an inlet port on the MS, and if a mixture or a whole cell (or organism) may further contain one or more analytes, which may comprise lipid, carbohydrate, nucleic acid and/or peptide structure or any other inorganic or organic structure. Samples may undergo treatments prior to MS, where the sample may be transformed to one in which, the MS-analyte is a derivative of the starting analyte, the amount(s) of non-analyte species have been changed compared to the starting sample, the relative occurrence of different MS-analytes in a sample is changed compared to the starting sample, the concentration of an MS-analyte is changed relative the corresponding starting analyte in the starting sample, or sample constituents, such as solvents, have been changed and/or the analyte has been changed from a dissolved form to a solid form, for instance in a co-crystallized form. Such treatments include, for example, digestion into fragments of various sizes and/or chemical derivatization of an analyte. Digestion may be purely chemical or enzymatic. Derivatization includes so-called mass tagging of either the starting analyte or of a fragment or other derivative formed during a sample treatment protocol. Other treatments include purifying and/or concentrating the sample prior to analysis. Such treatments apply, for example, to analytes that are biopolymers comprising carbohydrate, lipid, nucleic acid and/or peptide structure. Alternatively, the sample may also pass through the microchannel structure without being changed.

[00052] EXAMPLE ONE

[00053] The following example details the use of APCs as biosensors for disease. Reference standards of DC exposed to various pathogens are created, which are used in subsequent patient assays to determine exposure and to qualitate the immunological response to the pathogen.

[00054] Gene expression analysis: DC and Macrophages/monocytes from various donors are cultured in the presence and absence of pathogens, to initiate a response to the pathogen, then harvested. Total RNA is extracted from uninfected and infected cells, and used to create a reference array. Alternatively, the total RNA is converted to cDNA, before being fabricated into the reference array. Patient derived samples of DC are recovered, the nucleic acids extracted, and hybridized to the microarray then scanned. In the microarray data processing we use a filtering approach based on a fix change in average difference intensity values. For analysis of the raw data we use GenNet, an extremely robust expression data analysis platform. Using such a platform meets high-throughput demands, and is scalable.

[00055] Pathogen Genotyping: Since DC and Macrophages serve as pathogen reservoirs, enrichment of these cells and the use of genotypic analysis for the presence of the pathogen provides a novel screening method. Conventional methods of viral and bacterial disease diagnosis require the detection of the pathogens themselves, e.g., by blood cultures, or detection of pathogen-specific proteins or DNA/RNA, e.g., by PCR, and correlation of these findings with clinical symptoms. To overcome the limitations associated with conventional screening methods, we have developed high throughput genotyping assays, which are used to screen large numbers of pathogens. In addition, these assays are capable of detecting molecular variation in microbial strains; thus allowing distinction of, for example, route of transmission, origin, and relationship of a particular bacterial, viral, fungal, or parasitic strain, etc.

[00056] Mass Spectrometry (MS): To improve the success and productivity of peptide identification we have used the SELDI and MALDI time of flight (TOF) mass spectrometers. These are the most commonly used mass spectrometry methods for detecting peptide mass fingerprinting. This MALDI-O-TOF system uses orthogonal injection to introduce sample ions from the MALDI sources into a reflection TOF mass spectrometer. The MALDI sources of a conventional axial MALDI-TOF systems (linear or reflection mode) and is directly linked to the TOFMS. This direct linkage affects the instruments accuracy, resolution, and sensitivity because any discrepancies associated with the sample target are transferred to the detector. In contrast, using orthogonal geometry, the MALDI source is separated from the TOF, thus, eliminating discrepancies, increasing performance and simplifying method

development. The protein signatures found in untreated cultured compare to treated cultures are established.

[00057] Bio-marker detection: To detect bio-markers, we use commercially available pattern recognition and discover software from Eclipse Diagnostics, or similar software. This software allows for the rapid detection of genomic and proteonomic bio-markers and other complex biological relationships. These biomarkers are part of the database and are used as a pathogen-specific reference.

[00058] Advantages First, myeloid cells concentrate pathogens within the cell; thus, improved sensitivity of detection. Second, cDNA microarray technology is a high resolution technology capable of analyzing 52,000 or greater genes per sample and is independent of culturing the pathogen from blood. Our method of microarray construction involves a longer 70mer probe design and 30-fold internal redundancy per gene. Third, the ability to diagnose before symptoms occurs. In the case of bioengineered pathogens, traditional microbiology, ELISA or PCR based technology has to be created to detect new the new pathogen. By evaluating key cellular pathways (e.g. apoptotic, inflammatory, NFkB, and inflammatory mediators), we can detect early events in exposure. We have developed a method of rapid enrichment (1.5 h) of DC and Macrophages from the blood, which requires does not require prolonged culturing, or use of costly cytokines to force differentiation. In contrast, contemporary methods require culturing DC precursors for 7 days. DC in culture with prolonged exposure to cytokines is known to induce specific alteration in cellular pathways, have been demonstrated to modify pathogen infectivity, gene activation, and protein synthesis. High-throughput (HT) gene arrays allow the analysis of greater than 1,000 samples in per day. The number samples processed per day is equipment dependent which is also the case for the proteonomic technology (e.g. 10,000 samples/day). The integration of the HT system with the genomic and proteonomic database improves detection efficiency and also allows the real-time monitoring of progression of disease. Collectively, the combination serves to provide highly complex genomic- and proteonomic -based arrays and information databases for hospitals and laboratories, that can be shared in real time over networks.

[00059] The present invention provides for methods of rapidly identifying pathogens in the body and in the environment. The following pathogens are amenable

to detection and characterization using APC and the techniques described herein: bacteria, viruses, fungi, prions and protozoa.

[00060] Representative bacteria that are presented to APC to create pathogen exposed APC signatures include, Gram positive and Gram negative bacteria such as Staphylococcus, such as S. epidermis and S. aureus; Micrococcus; Streptococcus, such as S. pyogenes, S. equis, S. zooepidemicus, S. equisimilis, S. pneumoniae and S. agalactiae; Corynebacterium, such as C. pyogenes and C. pseudotuberculosis; Erysipelothrix such as E. rhusiopathiae; Listeria, such as L. monocytogenes; Bacillus, such as B. anthracis; Clostridium, such as C. perfringens; and Mycobacterium, such as M. tuberculosis and M. leprae. Gram negative bacterial species are exemplified by, but not limited to genera including: Escherichia, such as E. coli 0157:H7; Salmonella, such as S. typhi and S. gallinarum; Shigella, such as S. 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. aeruginosa; Klebsiella, such as K. pneumoniae; Pasteurella, such as P. multocida; Moraxella, such as M. bovis; Serratia, such as S. marcescens; Hemophilus, such as H. influenza; and Campylobacter species. Other species suitable for assays of the present invention include spirochetes such as those causing Lyme Disease, Enterococcus, Neisseria, Mycoplasma, Chlamidia, Francisella, Pasteurella, Brucella, and Enterobacteriaceae. Also detectable are CDC biological pathogens A, B, and C biological pathogens. Further examples of pathogenic bacterial species that are detectable according to the invention are obtained by reference to standard taxonomic and descriptive works such as Bergey's Manual of Determinative Bacteriology, 9th Ed., 1994, Williams and Wilkins, Baltimore, Md.

[00061] Representative viruses that are presented to APC to create pathogen exposed APC signatures include, adenovirus (such as can be found in infantile gastroenteritis, acute hemorrhagic cystitis, non-bacterial pneumonia, and viral conjunctivitis), herpesvirus (such as herpes simplex type I and type II, varicella zoster (the etiological agent of chicken pox), cytomegalovirus, and mononucleosis (the etiological agent Epstein-Barr virus)), poxvirus (the etiological agent for such disorders as smallpox (variola major and variola minor), Hepatitis A, B, and C, vaccinia virus, hantavirus and molluscum contagiosum), picornavirus (such as

rhinovirus (the common cold, also caused by coronavirus)) poliovirus (poliomyelitus)), an orthomyxovirus or paramyxovirus (such as influenza, and respiratory syncytial virus (RS)), parainfluenza virus (including such diseases as mumps), and rubeola (measles), rhabdovirus (rabies), vesicular stomatitis (VSV), togavirus such as rubella— the etiological agent causing German measles, and togaviridae causing encephalitis (EEE, WEE, and VEE), flavivirus such as the etiological agent causing Dengue Fever, West Nile Fever, Yellow Fever, and encephalitis, bunyavirus and arenavirus, reovirus, coronavirus such as the agent causing SARS, hepatitis, a papovavirus infection such as papilloma virus, a retroviral infection such as HIV, HTLV-I, and HTLV-II.

[00062] Representative fungi that are presented to APC to create pathogen exposed APC signatures include for example, Candida, such as C. albicans; Cryptococcus, such as C. neoformans; Malassezia (Pityrosporum); Histoplasma, such as H. capsulatum; Coccidioides, such as C. immitis; Hyphomyces, such as H. destruens; Blastomyces, such as B. dermatiditis; Aspergillus, such as A. fumigatus; Penicillium, such as P. marneffei; Pseudallescheria; Fusarium; Paecilomyces; Mucor/Rhizopus; and Pneumocystis, such as P. carinii. Subcutaneous fungi, such as species of Rhinosporidium and Sporothrix, and dermatophytes, such as Microsporum and Trichophyton species, are amenable to prevention and treatment by embodiments of the invention herein. Other disease causing fungi that can be detected include Trichophyton, Microsporum; Epidermophyton; Basidiobolus; Conidiobolus; Rhizopus Cunninghamelia; Rhizomucor; Paracoccidioides; Pseudallescheria; Rhinosporidium; and Sporothrix.

[00063] Representative protozoa that are presented to APC to create pathogen exposed APC signatures include the one or more single-celled, usually microscopic, eukaryotic organisms, such as amoebas, ciliates, flagellates, and sporozoans, for example, Plasmodium, Trypanosoma or Cryptosporidium.

[00064] EXAMPLE TWO

[00065] The generation of antigen presenting cell (APC)-specific signatures is a two step process. The first step involves obtaining a population of immune cells, and the fractionation of cell membranes and the enrichment of proteins from the

membrane, cytoplasm, and nucleus. Preferred cells are of the myeloid lineage, but PBMCs are suitable. Methods of enriching for myeloid cell populations, including DC, is described in US patents 6,589,526 and 6,194,204. Myeloid cells include monocytes and dendritic cells, in roughly 90% to 10% proportions. Antigenic markers for monocytes include CD 14+, HLA-DR or MHC class II, CD80+ CD86+. Antigenic markers for DC include CD2+, CD5+, CD 14+ CD83+ and CD90+. These are obtained by positive or negative selection methods. Preferred cell types are myeloid, which express antigenic markers consistent with both DC and monocyte cells. It is currently preferred to use freshly isolated, i.e., blood purified myeloid cells instead of cultured myeloid cells.

[00066] The following is a suggested procedure for isolation of monocytes from PBMC: Buffy coats were isolated from healthy volunteers (Transfusion Therapy, Children's Hospital, Boston, MA) and washed and concentrated with PBS. The buffy concentrate was then incubated with a modified monocyte enrichment means, such as the RosetteSep Kit, commercially available from StemCell Technologies, for example. This rosette cocktail contains anti-CD3, anti-CD 19, anti- CD54, and anti-CD62 monoclonal antibodies, which bind to T cells, B cells, NK cells and granulocytes. After 30 minute of incubation, this population was layered over ficoll gradient and centrifuged (Sorvall RT 6000, DuPont, Wilmington, DE) at 2500 rpm for 30 min to separate the low density DC and Mo from the high density (T, B, granulocytes and NK cells) density fractions. The low density cell population was >95% CD14 ^high by flow cytometry. These cells were incubated with a 1:100 dilution of mouse mAb (in ascitic fluid) to human CD2 (101d2-4Cl (anti-Tl 12); Dana-Farber Cancer Institute, Boston, MA) (26) for 30 min at 4°C, 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 the CD2+ cells, which were >96% pure, while the CD2- cells were >95% pure by flow cytometry with anti-CD2 and anti- CD 14. A blocking buffer containing 10% v/v heat-inactivated pooled human serum (PHS) (Nabi, Boca Raton, FL) and human IgG (50 mg/ml; Immuno AG, Vienna, Austria) in HBSS without magnesium and calcium (Cellgro; Fisher Scientific, Pittsburgh, PA) was used to prevent nonspecific mAb binding during each stage of isolation or flow cytometric analysis. For morphologic and functional studies of

freshly isolated, noncytokine-incubated CD2+ and CD2- Mo, we used culture medium (CM) containing RPMI 1640 (Cellgro) supplemented with 10% heat-inactivated PHS, 20 μg/ml gentamicin, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Gaithersburg, MD).

[00067] The second step involves obtaining mass spectra from the DC, for example using QSTAR (ABI), SELDI TOF from Ciphergen or proTOF from Perkin Elmer. Spectra are taken for naϊve APCs and those exposed to pathogens, tumors, or other antigens. In this example, we describe the process for obtaining individual data sets (signatures) from pathogen- APC or pathogen-food samples (i.e. Listeria-APC, Listeria-milk) to create a profile for bacterially contaminated and uncontaminated milk. The signature of a Listeria infected individual, obtained from sampling of APCs from that individual, is also provided.

[00068] Each profile includes a proteomic signature of, for example but not limited to, the cell membrane, cytoplasmic proteins, and nuclear protein characteristics, protein charges (i.e. positive and negative), Cu2+ chelating properties, cleavage patterns of native or denatured proteins with various endopeptidase, and the like, of the agents under study, measured by such properties as m/z size (kD), m/z intensity (uAmps), and standard deviation quantities. The frequency of occurrence of identifying features in a signature is corroborated by obtaining spectra of replicate samples, preferably 3-4 samples, thereby providing a consensus signature.

[00069] Several commonly used methods for isolating, fractionating or enriching sample proteins are as follows, others are known in the art. PARIS Kit - this is a kit commercial available from Ambion, and allows for the rapid isolation of RNA and proteins from samples. This kit may be used for those studies involving the isolation of APC proteins from APC-viruses or bacteria cocultures. For more sensitive detection and characterization of samples, it is advantageous to use cellular membrane fractions, which are obtained by common molecular biology techniques. The combination of the membrane, cytoplasm, and nuclear proteins enhances the sensitivity of APC-based detection methods.

[00070] Bacterial lysis by sonication can be used for both Gram-negative and

Gram-positive bacteria and uses sonication and optionally uses detergents, such as

Tween, Triton X-100, digitonin, CHAPS, SDS, Nonidet and others, which are highly recommended for profiles of Gram-negative bacteria or microorganisms with a thick or tough cell membrane. This protocol was used in the milk studies shown in FIG. 11-13. Harvest bacteria from agar plate into 5 ml of TEN Buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 100 mM NaCl). Pellet bacteria at 5,000 x g for 10 minutes. Subject the sample to 3 rounds of freeze/thawing in a dry ice/ethanol bath, thawing at 37°C. Resuspend the bacterial pellet into 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). Sonicate the suspension with 15 seconds burst followed by 30 seconds incubation on ice (4 rounds of sonication). Pellet bacterial debris at 14,000 rpm. Remove supernatant to fresh tube. Measure protein concentration by Bradford protein assay, standard curve, UV absorbtion or other method. Store at -70°C until ready for SELDI analysis.

[00071] Repeat freeze thawing involves repeated freeze thaw cycles to shear the cells. Sample were frozen in a dry ice-ethanol mixture and thawed at 37 degrees C. These steps were repeated 4-times and samples were spun in a microfuge at 14,000 rpm for 5 minutes producing a clear supernatant. The supernatant was removed and stored at -70 degrees C prior to sample runs. Bacterial lysis by French Press can be used for Gram-negative bacteria and is not recommended for Gram- positive bacteria. To perform this technique, resuspend pellets of bacteria in 20 mM HEPES pH 7.4, 50 mM NaCl, 1% Triton-X 100, 1 mM PMSF. Disrupt using a French Press at 750 psi. Remove cell debris by centrifugation at 20,000 x g for 20 min at 4 ⁰ C. Measure protein concentration in the supernatant by Bradford protein assay or similar assay. Store in aliquots at -7O ⁰ C until performing the SELDI technique. BugBuster Extraction kit- this is a commercially available kit sold by Novagen, which allows for gentle disruption of the cell wall of E. coli to release active proteins. This is a simple, rapid, low-cost alternative to French Press or sonication for releasing expressed target protein in a cell preparation. Alternatively, a buffer such as 10 mM Tris-HCl pH 7.4, 8 M Urea, 2% (w/v) CHAPS, ImM PMSF can also be used as lysis buffer. Store in aliquots at -7O ⁰ C until performing the SELDI technique.

[00072] The SELDI experimental protocol described below uses the IMAC

ProteinChip Array (PCA). The IMAC Arrays are coated with an NTA functional group to entrap transitional metals for subsequent metal affinity binding proteins. In these profiling studies, arrays are charged with copper prior to applying sample to the surface. Selectivity is determined by concentration of imidazole in the binding buffer. Increasing concentrations of imidazole in the binding/washing buffer, reduces the binding of protein with weaker affinities for metal, thereby reducing background signals. The protocol for IMAC PCA is described in detail below and is similar to the other Ciphergen PCA protocols.

[00073] Using the 8 spot arrays: Assemble the PCAs in the bioprocessor and add 50 microliters of IMAC charging solution to each well. Vortex for 5 minutes at RT. Remove the buffer from the wells. Rinse with water. Add 50 microliters of IMAC neutralization buffer to each well. Vortex for 5 minutes RT. Remove the buffer from well and rinse. Add 150 microliters of the IMAC binding buffer to each well and vortex at RT for 5 minutes. Remove buffer. Repeat binding buffer wash steps twice. Next add 90 microliters of IMAC binding buffer and 10 microliters of sample and vortex for 30 minutes at RT. The ratio of IMAC binding buffer to sample concentration can vary depending of the desired protein concentration. Remove the sample and wash with IMAC binding buffer three-times, each wash requires a 5 minute agitation step. Once completed, rinse with de-ionized water, drain wells of the bioprocessors, and let air dry. Apply 1.0 microliters EAM (matrix) solution to each spot and let air dry. PCA are analyzed on the Ciphergen Chip reader.

[00074] By contrast, the Perkin-Elmer proTOF experimental protocol applies the isolated-protein sample directly to the MALDI surface. However, in this process the MALDI surface binds to everything in the sample (i.e. proteins and nonproteins). The manufacturer suggests cleaning up the samples, for example microscale protein purification using Millipore ZIP TIPS®, filtered ion exchange pipette tips capable of removing certain proteins, thereby reducing total protein levels and reducing the complexity of the sample mixture. However, this technique is sensitive enough such that the tip performance, variable from tip to tip, can impact the resultant signature.

[00075] The next step involves derivation of marker proteins from the APC interaction with the pathogen. Once the marker proteins have been isolated, the

proteins provide templates for the generation of diagnostic antibodies. These antibodies to the derived APC proteins can be used for immunological assays, e.g., attached to a multiplexer or fluorescent readers for diagnostic purposes.

[00076] EXAMPLE THREE

[00077] This experiment investigated the reproducibility of the APC derived signatures for DC exposed to bacterial and viral pathogens. Listeria monocytogenes was processed by the bacterial lysis and sonication methods described above and evaluated on metal-binding (FIG. 1-2) or hydrophobic surfaces (FIG. 3-4). Samples were processed in parallel and analyzed on different PCA chips on different chip analyzers. The results demonstrate excellent experimental reproducibility. Data is displayed in spectral (FIG. 1, FIG. 3) or gel (FIG. 2, FIG. 4) views.

[00078] FIG. 5 illustrates the cytoplasmic protein profiles (signatures) from untreated/uninfected myeloid cells (Mx, top panel, (DC) mixture of dendritic cells, middle panel and (Mo) monocytes, bottom panel), DC, and Mo. Differences are observed in the individual profiles of DC and Mo.

[00079] FIGS 6-11 illustrate the cytoplasmic protein profiles (signatures) of

Mx, Mo, and DC cultured in the presence of a control Adenovirus (FIGS 6, 8, and 10) or in the presence of adenovirus with a single gene substitution (FIGS 7, 9, and 11). The observed signatures show that APCs infected with wild-type and mutant adenovirus can be identified and distinguished using the present invention. Unique protein signatures can be obtained that can differentiate between viruses having one gene substitution/modification.

[00080] FIGS. 12-14 illustrate the protein profiles (signatures) obtained from nuclear protein extracts of Mx (FIG. 12), DC (FIG. 13) and Mo (FIG. 14) cocultured in the presence of Listeria monocytogenes. APCs cultured in the presence of other Gram positive and Gram negative bacteria also generate unique signatures that can be used to identify the microorganism cocultured with the APCs. The proteomic signature of Listeria alone, i.e., not cultured with APC (bottom panel of FIG. 15) is distinct from the three coculture signatures, suggesting that Listeria overgrowth did not occur thereby contaminating the APC cocultures.

[00081] FIGS. 16-17 illustrate the spectroscopic profiles (signatures) of either skim or whole milk with Listeria contamination. FIG. 15 represents the spectral view of the results while FIGS. 16-17 represent the gel-views. The results demonstrate the ability of the present invention to detect the presence of unique pathogens in milk and other food stuffs, independent of APC detection methods.

[00082] EXAMPLE FOUR

[00083] In addition to pathological agents, the diagnostic methods include the detection, diagnosis and staging of various cancers and genetic disorders. Cancers are detectable by numerous markers. Malignant cells often express antigens that are not found in normal cells; some of these antigens are found at the surface of the cell, for example CEA (chorioembryonic antigen) and differentially glycosylated (hypoglycosylated) MUC-I, are two well-known tumor associated antigens. MUC- l/DF-3 is overexpressed in the majority of human carcinomas, multiple myeloma, acute myelogenous leukemia, acute lymphoblastic leukemia, and follicular lymphoma among others. The antigen can initiate an HLA-restricted T cell response following presentation of the antigen by DC (see, Brossart et al., (2001) Cancer Res. Sept.:61(18):6846-50. Other proteins upregulated in cancer cells include vascular endothelial growth factor (VEGF), Her-2/neu and hepsin. Intercellular adhesion molecule- 1 (ICAM-I), vascular adhesion molecule- 1 (VCAM-I) and E-Selectin (ELAM-I) play an important role in the complex series of events associated with inflammatory responses associated with cancer and tumor suppression.

[00084] In addition to pathological agents, the diagnostic methods include the detection, diagnosis and staging of various tumors and neoplasms. The invention can detect any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites. For example, APCs are cultured with the following cancers, to produce APC signatures of cancer-exposed cells: astrocytomas, gliomas, ependymomas, osteosarcoma, Ewing's sarcoma, retinoblastoma, bladder cancer, small and non-small cell lung cancer, oat cell lung cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, vaginal cancer, ovarian cancer, cancers of the liver, acute lymphocytic leukemia, acute myelogenous leukemia, lymphoma, myeloma, basal cell carcinoma, melanoma, thyroid follicular cancer, bladder carcinoma, glioma,

myelodysplastic syndrome, testicular cancer, stomach cancer, esophageal cancer, laryngeal cancer, squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, urothelial carcinoma, breast cancer or prostate cancer.

[00085] APC, particularly CD2+ DC are cultured with primary or metastatic tumor cells obtained by biopsy, preferably cells taken from representative stages of tumor growth. Alternatively, DC are cultured with purified preparations of CEA (chorioembryonic antigen) or differentially glycosylated (hypoglycosylated) MUC-I, or other tumor associated antigens. The DC lysates are used to prepare cDNA which is then used to create arrays of DC reference standards for high throughput screens. Arrays are prepared for each cancer type listed above, and preferably include each stage of the particular cancer type.

[00086] EXAMPLE FIVE

[00087] Kits are developed for isolation of samples from subjects and from the environment.

[00088] For patient diagnostic uses, the kits include reagents and materials for obtaining and isolating blood samples from patients, as well as reagents and materials for enriching cells such as APCs, PBMCs and more preferably DCs, and for processing the cells into cytoplasmic, nuclear, or membrane fractions and optionally for processing larger proteins into smaller peptides.

[00089] Kits may further include chips or plates and reagents, appropriate for use with mass spectroscopy, such as those produced by Ciphergen for SELDI and Perkin Elmer for MALDI-O-TOF. The kits also include suitable instructions for use. In certain embodiments, the kits include one or more of the APC arrays mentioned above, e.g., for use in diagnosing and staging cancers, or for determining the agent of infection and progression of the infection, or for forensic analysis. Other kit components include controls such as reference proteins, used to calibrate the mass spectrometer. In still other embodiments, the kit includes albumin or high molecular weight proteins, and is used for enhancing the resolution of low molecular with proteins in the signatures (the albumin bump technique). Kits for use with patients are suitable for human and veterinary uses.

[00090] The following kits are provided herein: biodefense kits for identifying pathogens associated with bioweapons in the environment, and in exposed subjects; agricultural kits for sampling contamination of food dairy products and livestock; endocrine and metabolic kits for assessing endocrine and metabolic function in a subject; neurological kits for assessing degenerative changes; infectious disease kits for identifying pathogens in an exposed subject; prenatal kits for assessing fetal health; cancer kits for diagnosing and staging cancer progression in a subject and for monitoring chemotherapy regimens and disease progression; cardiovascular kits for detecting early signs of cardiac damage and ischemia and vessel occlusion; renal kits for detecting damage in the subject from i.e., contrast agents and chemotherapy drugs.

[00091] EQUIVALENTS

[00092] From the foregoing detailed description of the specific embodiments of the invention, it should be apparent that unique detection methodologies have been described. Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. For instance, the choice of spectrum, or the APC used in the detection process is believed to be matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein.

References

[00093] All U.S. Patents and other references cited herein are hereby incorporated herein by reference in their entirety.

[00094] Arad, G., Levy, R., & Kaempfer, R. 2002. Superantigen concomitantly induces ThI cytokine genes and the ability to shut off their expression on re-exposure to superantigen. Immunol.Lett, 82(1-2): 75-78.

[00095] Chapes, S. K., Beharka, A. A., Hart, M. E., Smeltzer, M. S., & Iandolo,

J. J. 1994. Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages. J Leukoc.Biol, 55(4): 523-529.

[00096] Chaussabel D, Semnani RT, McDowell MA, Sacks D, Sher A, Nutman

TB (2003): Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites. Blood 102:672-8 1.

[00097] Clemens, D. L., Lee, B. Y., & Horwitz, M. A. 2004. Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages. Infect. Immun., 72(6): 3204-3217.

[00098] Comer, J. E., Galindo, C. L., Chopra, A. K., & Peterson, J. W. 2005.

GeneChip analyses of global transcriptional responses of murine macrophages to the lethal toxin of Bacillus anthracis. Infect.Immun., 73(3): 1879-1885.

[00099] Crawford K, Stark A, Kitchens B, Sternheim K, Pantazopoulos V,

Triantafellow E, Wang Z, Vasir B, Larsen CE, Gabuzda D, Reinherz E, Alper CA (2003): CD2 engagement induces dendritic cell activation: implications for immune surveillance and T-cell activation. Blood 102: 1745-52. Jantsch J, Cheminay C, Chakravortty D, Lindig T, Hem J, Hensel M (2003): Intracellular activities of Salmonella enterica in murine dendritic cells. 5:933-45.

[000100] Deringer, J. R., Ely, R. J., Stauffacher, C. V., & Bohach, G. A. 1996. Subtype-specific interactions of type C staphylococcal enterotoxins with the T-cell receptor. MoI Microbiol, 22(3): 523-534.

[000101] Frandji, P., Tkaczyk, C, Oskeritzian, C, David, B., Desaymard, C, & Mecheri, S. 1996. Exogenous and endogenous antigens are differentially presented by mast cells to CD4+ T lymphocytes. Eur.J Immunol., 26(10): 2517-2528.

[000102] Hernychova, L., Kovarova, H., Macela, A., Kroca, M., Krocova, Z., & Stulik, J. 1997. Early consequences of macrophage-Francisella tularensis interaction under the influence of different genetic background in mice. Immunol.Lett, 57(1-3): 75-81.

[000103] Hofer 5, Rescigno M, Granucci F, Citterio 5, Francolini M, Ricciardi- Castagnoli P (2001): Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide. Microbes and Infection / Institut Pasteur 3:259-65.

[000104] LeClaire, R. D., KeIl, W. M., Sadik, R. A., Downs, M. B., & Parker, G. W. 1995. Regulation of staphylococcal enterotoxin B-elicited nitric oxide production by endothelial cells. Infect.Immun., 63(2): 539-546.

[000105] Lemay, S., Mao, C, & Singh, A. K. 1996. Cytokine gene expression in the MRL/lpr model of lupus nephritis. Kidney Int, 50(1): 85-93.

[000106] Mascarell, L. & Truffa-Bachi, P. 2004. T lymphocyte activation initiates the degradation of the CD62L encoding mRNA and increases the transcription of the corresponding gene. Immunol.Lett, 94(1-2): 115-122.

[000107] Moon, Y. & Pestka, J. J. 2003. Cyclooxygenase-2 mediates interleukin-6 upregulation by vomitoxin (deoxynivalenol) in vitro and in vivo. Toxicol. Appl Pharmacol, 187(2): 80-88.

[000108] Novellino, P. S., Trejo, Y. G., Beviacqua, M., Bordenave, R. H., & Rumi, L. S. 2000. Regulation of HLA-DR antigen in monocytes from colorectal cancer patients by in vitro treatment with human recombinant interferon-gamma. J Investig.Allergol.Clin Immunol., 10(2): 90-93.

[000109] Pulendran B, Kumar P, Cutler CW, Mohamadzadeh M, Van Dyke T, Banchereau J (2001): Lipopolysaccharides from distinct pathogens induce different

classes of immune responses in vivo. Journal of Immunology (Baltimore, Md. : 1950) 167:5067-76.

[000110] Rachlis, A., Watson, J. L., Lu, J., & McKay, D. M. 2002. Nitric oxide reduces bacterial superantigen-immune cell activation and consequent epithelial abnormalities. J Leukoc.Biol, 72(2): 339-346.

[00011 1] Schlueter, A. J., Krieg, A. M., de Vries, P, & Li, X. 2001. Type I interferon is the primary regulator of inducible Ly-6C expression on T cells. J Interferon Cytokine Res, 21(8): 621-629.

[000112] Schmitz, J. & Radbruch, A. 1992. Distinct antigen presenting cell- derived signals induce TH cell proliferation and expression of effector cytokines. Int Immunol., 4(1): 43-51.

[000113] Sundquist M, Rydstrom A, Wick MJ (2004): Immunity to Salmonella from a dendritic point of view. Cell Microbiol 6:1 -Ii. Appelmelk BJ, van_Die I, van Vliet SJ, Vandenbroucke_Grauls CM, Geijtenbeek TB, van Kooyk Y (2003): Cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3 -grabbing nonintegrin on dendritic cells. Journal of Immunology (Baltimore, Md.: 1950) 170:1635-9.

[000114] Szabo, G., Mandrekar, P., & Catalano, D. 1995. Inhibition of superantigen-induced T cell proliferation and monocyte IL-I beta, TNF-alpha, and IL- 6 production by acute ethanol treatment. J Leukoc.Biol, 58(3): 342-350.

[000115] Szebeni, J. & Sykes, M. 1996. CAG repeat mutation in the mouse IL-2 gene enables concurrent assessment of T- and B-cell-specific gene expression by northern analysis. Exp.Clin Immunogenet., 13(2): 117-119.

[000116] Takahashi, M., Takahashi, M., Shinohara, F., Takada, H., & Rikiishi, H. 2001. Effects of superantigen and lipopolysaccharide on induction of CD80 through apoptosis of human monocytes. Infect.Immun., 69(6): 3652-3657.

[000117] Thomassen, M. J., Ahmad, M., Barna, B. P., Antal, J., Wiedemann, H. P., Meeker, D. P., Klein, J., Bauer, L., Gibson, V., Andresen, S., & . 1991. Induction of cytokine messenger RNA and secretion in alveolar macrophages and blood

monocytes from patients with lung cancer receiving granulocyte-macrophage colony- stimulating factor therapy. Cancer Res, 51(3): 857-862.

[000118] Tucker, A. E., Salles, I. L, Voth, D. E., Ortiz-Leduc, W., Wang, H., Dozmorov, L, Centola, M., & Ballard, J. D. 2003. Decreased glycogen synthase kinase 3 -beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages. Cell Microbiol, 5(8): 523-532.

[000119] Wakita, H., Tokura, Y., Furukawa, F., & Takigawa, M. 1995. Staphylococcal enterotoxin B upregulates expression of ICAM-I molecules on IFN- gamma-treated keratinocytes and keratinocyte cell lines. J Invest Dermatol., 105(4): 536-542.

[000120] Wang, Y., Wu, T. R., Cai, S., Welte, T., & Chin, Y. E. 2000. Statl as a component of tumor necrosis factor alpha receptor 1 -TRADD signaling complex to inhibit NF-kappaB activation. MoI Cell Biol, 20(13): 4505-4512.

[000121] Wrighton, C. J., Hofer-Warbinek, R., Moll, T., Eytner, R., Bach, F. H., & de Martin, R. 1996. Inhibition of endothelial cell activation by adenovirus-mediated expression of I kappa B alpha, an inhibitor of the transcription factor NF-kappa B. J Exp.Med, 183(3): 1013-1022.

[000122] Yang, Y., Kim, D., & Fathman, C. G. 1998. Regulation of programmed cell death following T cell activation in vivo. Int Immunol., 10(2): 175- 183.

[000123] Zembala, M., Czupryna, A., Wieckiewicz, J., Jasinski, M., Pryjma, J., Ruggiero, L, Siedlar, M., & Popiela, T. 1993. Tumour-cell-induced production of tumour necrosis factor by monocytes of gastric cancer patients receiving BCG immunotherapy. Cancer Immunol.Immunother., 36(2): 127-132.

[000124] Zoja, C, Corna, D., Camozzi, D., Cattaneo, D., Rottoli, D., Batani, C, Zanchi, C, Abbate, M., & Remuzzi, G. 2002. How to fully protect the kidney in a severe model of progressive nephropathy: a multidrug approach. J Am Soc Nephrol, 13(12): 2898-2908.