Patent Publication Number: US-2021189497-A1

Title: Analytical methods and arrays for use in the same

Description:
FIELD OF THE INVENTION 
     The present invention relates to an in vitro method for identifying agents capable of inducing sensitization of human skin and arrays and analytical kits for use in such methods. 
     BACKGROUND 
     Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. It is commonly caused by immunological responses towards chemical haptens leading to substantial economic burden for society. Current tests for sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within, e.g. the pharmaceutical and cosmetic industries, have stimulated significant research efforts to develop alternative human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. 
     Allergic contact dermatitis (ACD) is a common inflammatory skin disease characterized by eczema and recurrent episodes of itching [1]. The disease affects a significant proportion of the population, with prevalence rates of 7.2% to 18.6% in Europe [2, 3], and the incidence is increasing due to repeated exposure to sensitizing chemicals. ACD is a type IV delayed-type hypersensitivity response caused mainly by reactive T helper 1 (Th1) and interferon (IFN)γ producing CD8 +  T cells at site of contact with small chemical haptens in previously exposed, and immunologically sensitized, individuals [4]. Dendritic cells (DC) in the epidermis initiate the immune reactions by responding to haptens bound to self-molecules and activating T cell-mediated immunity. 
     The REACH (Registration, Evaluation, and Authorisation of Chemicals) regulation requires that all new and existing chemicals within the European Union, involving approximately 30 000 chemicals, should be tested for hazardous effects [5]. As the identification of potential sensitizers currently requires animal testing, the REACH legislation will have a huge impact on the number of animals needed for testing. Further, the 7th Amendment to the Cosmetics Directive (76/768/EEC) posed a ban on animal tests for the majority of cosmetic ingredients for human use, to be in effect by 2009, with the exceptions of some tests by 2013. Thus, development of reliable in vitro alternatives to experimental animals for the assessment of sensitizing capacity of chemicals is urgent. To date, no non-animal replacements are available for identification of skin sensitizing chemicals, instead the preferred assay is the mouse Local Lymph Node Assay (LLNA) [6], followed by the Guinea pig maximization test (GPMT) [7]. An in vitro alternative to these animal models would preferably exhibit improved reliability, accuracy and importantly correlate to human reactivity. 
     Dendritic cells (DCs) play key roles in the immune response by bridging the essential connections between innate and adaptive immunity. They can, upon triggering, rapidly produce large amounts of mediators, which influence migration and activation of other cells at the site of inflammation, and selectively respond to various pathogens and environmental factors, by fine-tuning the cellular response through antigen-presentation. Thus, exploring and utilizing the immunological decision-making by DCs during stimulation with sensitizers, could serve as a potent test strategy for prediction of sensitization. 
     However, multifaceted phenotypes and specialized functions of different DC subpopulations, as well as their wide and scarce distribution, are complicating factors, which impede the employment of primary DCs as a test platform. Hence, there is a real need to establish accurate and reliable in vitro assays that also circumvent the problems associated with variability of and difficulty in obtaining DCs. 
     DISCLOSURE OF THE INVENTION 
     Thus, the development of assays based on the predictability of DC function should preferably rely on alternative cell types or mimics of in vivo DCs. For this purpose, a cell line with DC characteristics would be advantageous, as it constitutes a stable, reproducible and unlimited supply of cells. In terms of DC mimics, differentiated myelomonocytic MUTZ-3 cells are by far the preferred candidate [8]. MUTZ-3 is as an unlimited source of CD34 +  DC progenitors and it can acquire, upon cytokine stimulation, phenotypes similar to immature DCs or Langerhans-like DCs [9], present antigens through CD1d, MHC class I and II and induce specific T-cell proliferation [8]. MUTZ-3 also displays a mature transcriptional and phenotypic profile upon stimulation with inflammatory mediators [10]. 
     The present inventors have developed a novel test principle for prediction of skin sensitizers. It has surprisingly been found that skin sensitizers can be accurately identified/predicted using DC progenitor cells, such as MUTZ-3 cells, without further differentiation in a process whereby the cells are stimulated with a panel of sensitizing chemicals, non-sensitizing chemicals, and/or other controls (e.g. vehicle controls comprising diluent only, such as DMSO and/or distilled water). This was found to substantially simplify and improve the reproducibility of the procedure. 
     The transcriptional response to chemical stimulation was assessed with genome-wide profiling. From data analysis, a biomarker signature of 200 transcripts was identified which completely separated the transcriptional response induced by sensitizing chemicals vs. non-sensitizing chemicals and vehicle controls. Further, the potent predictive power of the signature was illustrated, using SVM and ROC curve analysis. The biomarker signature include transcripts involved in relevant biological pathways, such as DC maturation and cytokine responses, which may shed light on the molecular interactions involved in the process of sensitization. In conclusion, a biomarker signature with potent predictive power, which represents a compelling readout for an in vitro assay useful for the identification of human sensitizing chemicals has been identified. 
     Hence, a first aspect of the present invention provides an in vitro method for identifying agents capable of inducing sensitization of mammalian skin comprising or consisting of the steps of:
         a) exposing a population of dendritic cells or a population of dendritic-like cells to a test agent; and   b) measuring in the cells the expression of one or more biomarker(s) selected from the group defined in Table 3,       

     wherein the expression in the cells of the one or more biomarkers measured in step (b) is indicative of the sensitizing effect of the test agent. 
     By “agents capable of inducing sensitization of mammalian skin” we mean any agent capable of inducing and triggering a Type IV delayed-type hypersensitivity reaction in a mammal. Preferably, the Type IV delayed-type hypersensitivity reaction is DC-mediated. 
     In one embodiment, the “agents capable of inducing sensitization of mammalian skin” is an agent capable of inducing and triggering a Type IV delayed-type hypersensitivity reaction at a site of epidermal contact in a mammal. 
     The mammal may be any domestic or farm animal. Preferably, the mammal is a rat, mouse, guinea pig, cat, dog, horse or a primate. Most preferably, the mammal is human. As discussed above, in vivo methods of determining sensitisation are known in the art. A preferred method is the Local lymph node assay (for details, see Basketter, D. A., et al.,  Local lymph node assay—validation, conduct and use in practice.  Food Chem Toxicol, 2002. 40(5): p. 593-8). A further suitable, but less preferred, method is the guinea pig maximization test (for details, see Magnusson, B. and A. M. Kligman,  The identification of contact allergens by animal assay. The guinea pig maximization test.  J Invest Dermatol, 1969. 52(3): p. 268-76). 
     By “dendritic-like cells” we mean non-dendritic cells that exhibit functional and phenotypic characteristics specific to dendritic cells such as morphological characteristics, expression of costimulatory molecules and MHC class II molecules, and the ability to pinocytose macromolecules and to activate resting T cells. 
     In one embodiment, the dendritic-like cells are CD34 +  dendritic cell progenitors. Optionally, the CD34 +  dendritic cell progenitors can acquire, upon cytokine stimulation, the phenotypes of presenting antigens through CD1d, MHC class I and II, induce specific T-cell proliferation, and/or displaying a mature transcriptional and phenotypic profile upon stimulation with inflammatory mediators (i.e. similar phenotypes to immature dendritic cells or Langerhans-like dendritic cells). 
     Dendritic cells may be recognized by function, by phenotype and/or by gene expression pattern, particularly by cell surface phenotype. These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression and ability to present antigen to CD4+ and/or CD8+ T cells, particularly to naïve T cells (Steinman et al. (1991) Ann. Rev. Immunol. 9: 271). 
     The cell surface of dendritic cells is unusual, with characteristic veil-like projections, and is characterized by expression of the cell surface markers CD11c and MHC class II. Most DCs are negative for markers of other leukocyte lineages, including T cells, B cells, monocytes/macrophages, and granulocytes. Subpopulations of dendritic cells may also express additional markers including 33D1, CCR1, CCR2, CCR4, CCR5, CCR6, CCR7, CD1a-d, CD4, CD5, CD8alpha, CD9, CD11b, CD24, CD40, CD48, CD54, CD58, CD80, CD83, CD86, CD91, CD117, CD123 (IL3Ra), CD134, CD137, CD150, CD153, CD162, CXCR1, CXCR2, CXCR4, DCIR, DC-LAMP, DC-SIGN, DEC205, E-cadherin, Langerin, Mannose receptor, MARCO, TLR2, TLR3 TLR4, TLR5, TLR6, TLR9, and several lectins. 
     The patterns of expression of these cell surface markers may vary along with the maturity of the dendritic cells, their tissue of origin, and/or their species of origin. Immature dendritic cells express low levels of MHC class II, but are capable of endocytosing antigenic proteins and processing them for presentation in a complex with MHC class II molecules. Activated dendritic cells express high levels of MHC class 11, ICAM-1 and CD86, and are capable of stimulating the proliferation of naive allogeneic T cells, e. g. in a mixed leukocyte reaction (MLR). 
     Functionally, dendritic cells or dendritic-like cells may be identified by any convenient assay for determination of antigen presentation. Such assays may include testing the ability to stimulate antigen-primed and/or naive T cells by presentation of a test antigen, followed by determination of T cell proliferation, release of IL-2, and the like. 
     By “expression” we mean the level or amount of a gene product such as mRNA or protein. 
     Methods of detecting and/or measuring the concentration of protein and/or nucleic acid are well known to those skilled in the art, see for example Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press. 
     Preferred methods for detection and/or measurement of protein include Western blot, North-Western blot, immunosorbent assays (ELISA), antibody microarray, tissue microarray (TMA), immunoprecipitation, in situ hybridisation and other immunohistochemistry techniques, radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference. Antibody staining of cells on slides may be used in methods well known in cytology laboratory diagnostic tests, as well known to those skilled in the art. 
     Typically, ELISA involves the use of enzymes which give a coloured reaction product, usually in solid phase assays. Enzymes such as horseradish peroxidase and phosphatase have been widely employed. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from  Escherichia coli  provides a good conjugate because the enzyme is not present in tissues, is stable and gives a good reaction colour. Chemi-luminescent systems based on enzymes such as luciferase can also be used. 
     Conjugation with the vitamin biotin is frequently used since this can readily be detected by its reaction with enzyme-linked avidin or streptavidin to which it binds with great specificity and affinity. 
     Preferred methods for detection and/or measurement of nucleic acid (e.g. mRNA) include southern blot, northern blot, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography and in situ hybridisation. 
     In one embodiment the method comprises exposing a separate population of the dendritic cells or dendritic-like cells to a negative control agent that does not sensitize human skin, and measuring in the cells the expression of the one or more biomarker(s) measured in step (b). Hence, a sensitizing effect of the test agent is indicated in the event that the expression in the cell population of the one or more biomarker(s) measured in step (b) is/are different from the expression in the negative control sample. 
     In one embodiment, the negative control agent is a solvent for use with the test or control agents of the invention. Hence, the negative control may be DMSO and/or distilled water. 
     In another embodiment, the expression of one or more biomarkers measured in step (b) of the dendritic cells or dendritic-like cells prior to test agent exposure is used as a negative control. 
     A further embodiment comprises exposing a separate population of the dendritic cells or dendritic-like cells to a positive control agent that sensitizes human skin and measuring in the cells the expression of the one or more biomarker(s) measured in step (b). Hence, a sensitizing effect of the test agent is indicated in the event that the expression in the cell population of the one or more biomarker(s) measured in step (b) is/are similar to or the same as the expression in the positive control sample. 
     Preferably the method comprises, in step (b), measuring the expression of at least one biomarker selected from the group consisting of:
         i) taste receptor, type 2, member 5 (TAS2R5),   ii) keratinocyte growth factor-like protein 1/2/hypothetical protein FLJ20444 (KGFLP1/2/FLJ20444),   iii) transmembrane anterior posterior transformation 1 (TAPT1),   iv) sprouty homolog 2 (SPRY2),   v) fatty acid synthase (FASN),   vi) B-cell CLL/lymphoma 7A (BCL7A),   vii) solute carrier family 25, member 32 (SLC25A32),   viii) ferritin, heavy polypeptide pseudogene 1 (FTHP1),   ix) ATPase, H+ transporting, lysosomal 50/57 kDa, V1 subunit H (ATP6V1H),   x) squalene epoxidase (SQLE), and   xi) histone cluster 1, H1e (HIST1H1E).       

     Hence, in one embodiment the expression of taste receptor, type 2, member 5 (TAS2R5) is measured in step (b). In a further embodiment, in step (b), the expression of keratinocyte growth factor-like protein 1/2/hypothetical protein FLJ20444 (KGFLP1/2/FLJ20444) is measured. The method may comprise measuring the expression of transmembrane anterior posterior transformation 1 (TAPT1) in step (b). In one embodiment, the method comprises measuring the expression of sprouty homolog 2 (SPRY2) in step (b). However, a further embodiment the method, in step (b), comprises measuring the expression of fatty acid synthase (FASN). The method may comprise measuring the expression of B-cell CLL/lymphoma 7A (BCL7A) in step (b). It may also comprise measuring the expression of solute carrier family 25, member 32 (SLC25A32) in step (b). It may additionally comprise, in step (b), measuring the expression of ferritin, heavy polypeptide pseudogene 1 (FTHP1). A still further embodiment comprises measuring the expression of ATPase, H+transporting, lysosomal 50/57 kDa, V1 subunit H (ATP6V1H) in step (b). In another embodiment step (b) comprises measuring the expression of squalene epoxidase (SQLE). In yet another embodiment the expression of histone cluster 1, H1e (HIST1H1E) is measured in step (b). 
     The method may comprise or consist of measuring, in step (b), the expression of at least 2 biomarkers from Table 3A, for example, at least 3, 4, 5, 6, 7, 8, 9, 10 or 11 biomarkers from Table 3A. In a preferred embodiment, the method comprises or consists of measuring the expression of fatty acid synthase (FASN) and squalene epoxidase (SQLE) in step (b). 
     The method may additionally or alternatively comprise or consist of, measuring in step (b) the expression of at least 2 biomarkers from Table 3B, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, or at least 189 biomarkers from Table 3B. 
     Thus, the expression of all of the biomarkers in Table 3A and/or all of the biomarkers in Table 3B may be measured in step (b). 
     In a preferred embodiment, step (b) comprises or consists of measuring the expression of a nucleic acid molecule encoding the one or more biomarker(s). The nucleic acid molecule may be a cDNA molecule or an mRNA molecule. Preferably, the nucleic acid molecule is an mRNA molecule. 
     In one embodiment the expression of the one or more biomarker(s) in step (b) is performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography and in situ hybridisation. Preferably, the expression of the one or more biomarker(s) is measured using a DNA microarray. 
     The method may comprise measuring the expression of the one or more biomarker(s) in step (b) using one or more binding moieties, each capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table 3. In one embodiment the one or more binding moieties each comprise or consist of a nucleic acid molecule. In a further embodiment the one or more binding moieties each comprise or consist of DNA, RNA, PNA, LNA, GNA, TNA or PMO. Preferably, the one or more binding moieties each comprise or consist of DNA. In one embodiment, the one or more binding moieties are 5 to 100 nucleotides in length. However, in an alternative embodiment, they are 15 to 35 nucleotides in length. 
     Suitable binding agents (also referred to as binding molecules) may be selected or screened from a library based on their ability to bind a given nucleic acid, protein or amino acid motif, as discussed below. 
     In a preferred embodiment, the binding moiety comprises a detectable moiety. 
     By a “detectable moiety” we include a moiety which permits its presence and/or relative amount and/or location (for example, the location on an array) to be determined, either directly or indirectly. 
     Suitable detectable moieties are well known in the art. 
     For example, the detectable moiety may be a fluorescent and/or luminescent and/or chemiluminescent moiety which, when exposed to specific conditions, may be detected. Such a fluorescent moiety may need to be exposed to radiation (i.e. light) at a specific wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at a specific wavelength that may be detected. 
     Alternatively, the detectable moiety may be an enzyme which is capable of converting a (preferably undetectable) substrate into a detectable product that can be visualised and/or detected. Examples of suitable enzymes are discussed in more detail below in relation to, for example, ELISA assays. 
     Hence, the detectable moiety may be selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety (for example, a radioactive atom); or an enzymatic moiety. Preferably, the detectable moiety comprises or consists of a radioactive atom. The radioactive atom may be selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90. 
     Clearly, the agent to be detected (such as, for example, the one or more biomarkers in the test sample and/or control sample described herein and/or an antibody molecule for use in detecting a selected protein) must have sufficient of the appropriate atomic isotopes in order for the detectable moiety to be readily detectable. 
     In an alternative preferred embodiment, the detectable moiety of the binding moiety is a fluorescent moiety. 
     The radio- or other labels may be incorporated into the biomarkers present in the samples of the methods of the invention and/or the binding moieties of the invention in known ways. For example, if the binding agent is a polypeptide it may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as  99m Tc,  123 I,  186 Rh,  188 Rh and  111 In can, for example, be attached via cysteine residues in the binding moiety. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978)  Biochem. Biophys. Res. Comm.  80, 49-57) can be used to incorporate  123 I. Reference (“Monoclonal Antibodies in Immunoscintigraphy”, J-F Chatal, CRC Press, 1989) describes other methods in detail. Methods for conjugating other detectable moieties (such as enzymatic, fluorescent, luminescent, chemiluminescent or radioactive moieties) to proteins are well known in the art. 
     It will be appreciated by persons skilled in the art that biomarkers in the sample(s) to be tested may be labelled with a moiety which indirectly assists with determining the presence, amount and/or location of said proteins. Thus, the moiety may constitute one component of a multicomponent detectable moiety. For example, the biomarkers in the sample(s) to be tested may be labelled with biotin, which allows their subsequent detection using streptavidin fused or otherwise joined to a detectable label. 
     In another embodiment of first aspect of the present invention step (b) comprises determining the expression of the protein of the one or more biomarker(s). The method may comprise measuring the expression of the one or more biomarker(s) in step (b) using one or more binding moieties each capable of binding selectively to one of the biomarkers identified in Table 3. The one or more binding moieties may comprise or consist of an antibody or an antigen-binding fragment thereof such as a monoclonal antibody or fragment thereof. 
     The term “antibody” includes any synthetic antibodies, recombinant antibodies or antibody hybrids, such as but not limited to, a single-chain antibody molecule produced by phage-display of immunoglobulin light and/or heavy chain variable and/or constant regions, or other immunointeractive molecules capable of binding to an antigen in an immunoassay format that is known to those skilled in the art. 
     We also include the use of antibody-like binding agents, such as affibodies and aptamers. 
     A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter &amp; Milstein (1991)  Nature  349, 293-299. 
     Additionally, or alternatively, one or more of the first binding molecules may be an aptamer (see Collett et al., 2005,  Methods  37:4-15). 
     Molecular libraries such as antibody libraries (Clackson et al, 1991,  Nature  352, 624-628; Marks et al, 1991,  J Mol Biol  222(3): 581-97), peptide libraries (Smith, 1985,  Science  228(4705): 1315-7), expressed cDNA libraries (Santi et al (2000)  J Mol Biol  296(2): 497-508), libraries on other scaffolds than the antibody framework such as affibodies (Gunneriusson et al, 1999,  Appl Environ Microbiol  65(9): 4134-40) or libraries based on aptamers (Kenan et al, 1999,  Methods Mol Biol  118, 217-31) may be used as a source from which binding molecules that are specific for a given motif are selected for use in the methods of the invention. 
     The molecular libraries may be expressed in vivo in prokaryotic cells (Clackson et al, 1991, op. cit.; Marks et al, 1991, op. cit.) or eukaryotic cells (Kieke et al, 1999,  Proc Natl Acad Sci USA,  96(10):5651-6) or may be expressed in vitro without involvement of cells (Hanes &amp; Pluckthun, 1997,  Proc Natl Acad Sci USA  94(10):4937-42; He &amp; Taussig, 1997,  Nucleic Acids Res  25(24):5132-4; Nemoto et al, 1997,  FEBS Lett,  414(2):405-8). 
     In cases when protein based libraries are used, the genes encoding the libraries of potential binding molecules are often packaged in viruses and the potential binding molecule displayed at the surface of the virus (Clackson et al, 1991, supra; Marks et al, 1991, supra; Smith, 1985, supra). 
     Perhaps the most commonly used display system is filamentous bacteriophage displaying antibody fragments at their surfaces, the antibody fragments being expressed as a fusion to the minor coat protein of the bacteriophage (Clackson et al, 1991, supra; Marks et al, 1991, supra). However, other suitable systems for display include using other viruses (EP 39578), bacteria (Gunneriusson et al, 1999, supra; Daugherty et al, 1998,  Protein Eng  11(9):825-32; Daugherty et al, 1999,  Protein Eng  12(7):613-21), and yeast (Shusta et al, 1999,  J Mol Biol  292(5):949-56). 
     In addition, display systems have been developed utilising linkage of the polypeptide product to its encoding mRNA in so-called ribosome display systems (Hanes &amp; Pluckthun, 1997, supra; He &amp; Taussig, 1997, supra; Nemoto et al, 1997, supra), or alternatively linkage of the polypeptide product to the encoding DNA (see U.S. Pat. No. 5,856,090 and WO 98/37186). 
     The variable heavy (V H ) and variable light (V L ) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by “humanisation” of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984)  Proc. Natl. Acad. Sci. USA  81, 6851-6855). 
     That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988)  Science  240, 1041); Fv molecules (Skerra et al (1988)  Science  240, 1038); single-chain Fv (ScFv) molecules where the V H  and V L  partner domains are linked via a flexible oligopeptide (Bird et al (1988)  Science  242, 423; Huston et al (1988)  Proc. Natl. Acad. Sci. USA  85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989)  Nature  341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter &amp; Milstein (1991)  Nature  349, 293-299. 
     The antibody or antigen-binding fragment may be selected from the group consisting of intact antibodies, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′ fragments and F(ab) 2  fragments), single variable domains (e.g. V H  and V L  domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]). Preferably, the antibody or antigen-binding fragment is a single chain Fv (scFv). 
     The one or more binding moieties may alternatively comprise or consist of an antibody-like binding agent, for example an affibody or aptamer. 
     By “scFv molecules” we mean molecules wherein the V H  and V L  partner domains are linked via a flexible oligopeptide. 
     The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Effector functions of whole antibodies, such as complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from  E. coli,  thus allowing the facile production of large amounts of the said fragments. 
     Whole antibodies, and F(ab′) 2  fragments are “bivalent”. By “bivalent” we mean that the said antibodies and F(ab′) 2  fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites. 
     The antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and applications”, J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference. 
     When potential binding molecules are selected from libraries, one or more selector peptides having defined motifs are usually employed. Amino acid residues that provide structure, decreasing flexibility in the peptide or charged, polar or hydrophobic side chains allowing interaction with the binding molecule may be used in the design of motifs for selector peptides. For example:
         (i) Proline may stabilise a peptide structure as its side chain is bound both to the alpha carbon as well as the nitrogen;   (ii) Phenylalanine, tyrosine and tryptophan have aromatic side chains and are highly hydrophobic, whereas leucine and isoleucine have aliphatic side chains and are also hydrophobic;   (iii) Lysine, arginine and histidine have basic side chains and will be positively charged at neutral pH, whereas aspartate and glutamate have acidic side chains and will be negatively charged at neutral pH;   (iv) Asparagine and glutamine are neutral at neutral pH but contain a amide group which may participate in hydrogen bonds;   (v) Serine, threonine and tyrosine side chains contain hydroxyl groups, which may participate in hydrogen bonds.       

     Typically, selection of binding molecules may involve the use of array technologies and systems to analyse binding to spots corresponding to types of binding molecules. 
     The one or more protein-binding moieties may comprise a detectable moiety. The detectable moiety may be selected from the group consisting of a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety and an enzymatic moiety. 
     In a further embodiment of the methods of the invention, step (b) may be performed using an assay comprising a second binding agent capable of binding to the one or more proteins, the second binding agent also comprising a detectable moiety. Suitable second binding agents are described in detail above in relation to the first binding agents. 
     Thus, the proteins of interest in the sample to be tested may first be isolated and/or immobilised using the first binding agent, after which the presence and/or relative amount of said biomarkers may be determined using a second binding agent. 
     In one embodiment, the second binding agent is an antibody or antigen-binding fragment thereof; typically a recombinant antibody or fragment thereof. Conveniently, the antibody or fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule. Suitable antibodies and fragments, and methods for making the same, are described in detail above. 
     Alternatively, the second binding agent may be an antibody-like binding agent, such as an affibody or aptamer. 
     Alternatively, where the detectable moiety on the protein in the sample to be tested comprises or consists of a member of a specific binding pair (e.g. biotin), the second binding agent may comprise or consist of the complimentary member of the specific binding pair (e.g. streptavidin). 
     Where a detection assay is used, it is preferred that the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety. Examples of suitable detectable moieties for use in the methods of the invention are described above. 
     Preferred assays for detecting serum or plasma proteins include enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference. Antibody staining of cells on slides may be used in methods well known in cytology laboratory diagnostic tests, as well known to those skilled in the art. 
     Thus, in one embodiment the assay is an ELISA (Enzyme Linked Immunosorbent Assay) which typically involves the use of enzymes which give a coloured reaction product, usually in solid phase assays. Enzymes such as horseradish peroxidase and phosphatase have been widely employed. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from  Escherichia coli  provides a good conjugate because the enzyme is not present in tissues, is stable and gives a good reaction colour. Chemiluminescent systems based on enzymes such as luciferase can also be used. 
     Conjugation with the vitamin biotin is frequently used since this can readily be detected by its reaction with enzyme-linked avidin or streptavidin to which it binds with great specificity and affinity. 
     In an alternative embodiment, the assay used for protein detection is conveniently a fluorometric assay. Thus, the detectable moiety of the second binding agent may be a fluorescent moiety, such as an Alexa fluorophore (for example Alexa-647). 
     Preferably, step (b) is performed using an array. The array may be a bead-based array or a surface-based array. The array may be selected from the group consisting of: macroarray; microarray; nanoarray. 
     In on embodiment, the method is for identifying agents capable of inducing a hypersensitivity response in human skin. Preferably, the hypersensitivity response is a cell-mediated hypersensitivity response, for example, a type IV hypersensitivity response. Preferably, the method is for identifying agents capable of inducing allergic contact dermatitis (ACD) (i.e. the hypersensitivity response is ACD). 
     In one embodiment, the population of dendritic cells or population of dendritic-like cells is a population of dendritic cells. Preferably, the dendritic cells are primary dendritic cells. Preferably, the dendritic cells are myeloid dendritic cells. 
     The population of dendritic cells or dendritic-like cells is preferably mammalian in origin. Preferably, the mammal is a rat, mouse, guinea pig, cat, dog, horse or a primate. Most preferably, the mammal is human. 
     In an embodiment the population of dendritic cells or population of dendritic-like cells is a population of dendritic-like cells, preferably myeloid dendritic-like cells. 
     In one embodiment, the dendritic-like cells express at least one of the markers selected from the group consisting of CD54, CD86, CD80, HLA-DR, CD14, CD34 and CD1a, for example, 2, 3, 4, 5, 6 or 7 of the markers. In a further embodiment, the dendritic-like cells express the markers CD54, CD86, CD80, HLA-DR, CD14, CD34 and CD1a. 
     In a further embodiment, the dendritic-like cells may be derived from myeloid dendritic cells. Preferably the dendritic-like cells are myeloid leukaemia-derived cells. Preferably, the myeloid leukaemia-derived cells are selected from the group consisting of KG-1, THP-1, U-937, HL-60, Monomac-6, AML-193 and MUTZ-3. Most preferably, dendritic-like cells are MUTZ-3 cells. MUTZ-3 cells are human acute myelomonocytic leukemia cells that were deposited with Deutsche Sammlung für Mikroorganismen and Zellkulturen GmbH (DSMZ), (Inhoffensrasse 7B, Braunschweig, Germany on 15 May 1995 (www.dsmz.de; deposit number ACC 295). 
     In one embodiment, the dendritic-like cells, after stimulation with cytokine, present antigens through CD1d, MHC class I and II and/or induce specific T-cell proliferation. 
     In one embodiment, the negative control agent(s) is/are selected from the group consisting of 1-Butanol, 4-Aminobenzoic acid, Benzaldehyde, Chlorobenzene, Diethyl phthalate, Dimethyl formamide, Ethyl vanillin, Glycerol, Isopropanol, Lactic acid, Methyl salicylate, Octanoic acid, Propylene glycol, Phenol, p-ydroxybenzoic acid, Potassium permanganate, Salicylic acid, Sodium dodecyl sulphate, Tween 80 and Zinc sulphate. 
     The method may comprise the use of at least 2 negative control agents (i.e. non-sensitizing agents), for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 negative control agents. 
     In another embodiment, the positive control agent(s) is/are selected from the group consisting of 2,4-Dinitrochlorobenzene, Oxazolone, Potassium dichromate, Kathon CH (MC/MCI), Formaldehyde, 2-Aminophenol, 2-nitro-1,4-Phenylendiamine, p-Phenylendiamine, Hexylcinnamic aldehyde, 2-Hydroxyethyl acrylate, 2-Mercaptobenzothiazole, Glyoxal, Cinnamaldehyde, Isoeugenol, Ethylendiamine, Resorcinol, Cinnamic alcohol, Eugenol, Penicillin G or Geraniol. 
     The method may comprise the use of at least 2 positive control (i.e. sensitizing agents) are provided, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 positive control agents. 
     In one embodiment, the method is indicative of whether the test agent is or is not a sensitizing agent. In alternative or additional embodiment, the method is indicative of the sensitizing potency of the sample to be tested. 
     In one embodiment the method is indicative of the local lymph node assay (LLNA) classification of the sensitizing potency of the sample to be tested. For a detailed description of LLNA see Basketter, D. A., et al.,  Local lymph node assay—validation, conduct and use in practice.  Food Chem Toxicol, 2002. 40(5): p. 593-8 which is incorporated herein by reference. 
     In an alternative embodiment, the method is indicative of the guinea pig maximization test classification of the sensitizing potency of the sample to be tested. For a detailed description of the guinea pig maximization test see Magnusson, B. and A. M. Kligman,  The identification of contact allergens by animal assay. The guinea pig maximization test.  J Invest Dermatol, 1969. 52(3): p. 268-76, which is incorporated herein by reference. 
     Thus, in one embodiment, the method is indicative that the test agent is either, a non-sensitizer, a weak sensitizer, a moderate sensitizer, a strong sensitizer or an extreme sensitizer. The decision value and distance in PCA correlates with sensitizer potency. 
     Generally, skin sensitizing agents are determined with an ROC AUC of at least 0.55, for example with an ROC AUC of at least, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99 or with an ROC AUC of 1.00. Preferably, skin sensitizing agents are determined with an ROC AUC of at least 0.85, and most preferably with an ROC AUC of 1. 
     Typically, agents capable of inducing sensitization are identified using a support vector machine (SVM), such as those available from cran.r-project.org/web/packages/e1071/index.html (e.g. e1071 1.5-24). However, any other suitable means may also be used. 
     Support vector machines (SVMs) are a set of related supervised learning methods used for classification and regression. Given a set of training examples, each marked as belonging to one of two categories, an SVM training algorithm builds a model that predicts whether a new example falls into one category or the other. Intuitively, an SVM model is a representation of the examples as points in space, mapped so that the examples of the separate categories are divided by a clear gap that is as wide as possible. New examples are then mapped into that same space and predicted to belong to a category based on which side of the gap they fall on. 
     More formally, a support vector machine constructs a hyperplane or set of hyperplanes in a high or infinite dimensional space, which can be used for classification, regression or other tasks. Intuitively, a good separation is achieved by the hyperplane that has the largest distance to the nearest training datapoints of any class (so-called functional margin), since in general the larger the margin the lower the generalization error of the classifier. For more information on SVMs, see for example, Burges, 1998,  Data Mining and Knowledge Discovery,  2:121-167. 
     In one embodiment of the invention, the SVM is ‘trained’ prior to performing the methods of the invention using biomarker profiles of known agents (namely, known sensitizing or non-sensitizing agents). By running such training samples, the SVM is able to learn what biomarker profiles are associated with agents capable of inducing sensitization. Once the training process is complete, the SVM is then able whether or not the biomarker sample tested is from a sensitizing agent or a non-sensitizing agent. 
     This allows test agents to be classified as sensitizing or non-sensitizing. Moreover, by training the SVM with sensitizing agents of known potency (i.e. non-sensitizing, weak, moderate, strong or extreme sensitizing agents), the potency of test agents can also be identified comparatively. 
     However, this training procedure can be by-passed by pre-programming the SVM with the necessary training parameters. For example, agents capable of inducing sensitization be identified according to the known SVM parameters using the SVM algorithm detailed in Table 5, based on the measurement of all the biomarkers listed in Table 3(A) and 1(B). 
     It will be appreciated by skilled persons that suitable SVM parameters can be determined for any combination of the biomarkers listed Table 3 by training an SVM machine with the appropriate selection of data (i.e. biomarker measurements from cells exposed to known sensitizing and/or non-sensitizing agents). 
     Preferably, the method of the invention has an accuracy of at least 73%, for example 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% accuracy. 
     Preferably, the method of the invention has a sensitivity of at least 73%, for example 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sensitivity. 
     Preferably, the method of the invention has a specificity of at least 68%, for example 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% specificity. 
     By “accuracy” we mean the proportion of correct outcomes of a method, by “sensitivity” we mean the proportion of all positive chemicals that are correctly classified as positives, and by “specificity” we mean the proportion of all negative chemicals that are correctly classified as negatives. 
     A second aspect of the invention provides an array for use in the method of the first aspect of the invention (or any embodiment or combination of embodiments thereof), the array comprising one or more binding moieties as defined above. In one embodiment, the binding moieties are (collectively) capable of binding to all of the biomarkers defined in Table 3A. In a further embodiment, the binding moieties are (collectively) capable of binding to all of the biomarkers defined in Table 3B. Preferably, the binding moieties are (collectively) capable of binding to all of the biomarkers defined in Table 3A and Table 3B. 
     The binding moieties may be immobilised. 
     Arrays per se are well known in the art. Typically they are formed of a linear or two-dimensional structure having spaced apart (i.e. discrete) regions (“spots”), each having a finite area, formed on the surface of a solid support. An array can also be a bead structure where each bead can be identified by a molecular code or colour code or identified in a continuous flow. Analysis can also be performed sequentially where the sample is passed over a series of spots each adsorbing the class of molecules from the solution. The solid support is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane (e.g. plastic, polymer, perspex, silicon, amongst others), a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilising proteins, polynucleotides and other suitable molecules and/or conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing a protein molecule, polynucleotide or the like to the solid support. Alternatively, affinity coupling of the probes via affinity-tags or similar constructs may be employed. By using well-known techniques, such as contact or non-contact printing, masking or photolithography, the location of each spot can be defined. For reviews see Jenkins, R. E., Pennington, S. R. (2001,  Proteomics,  2,13-29) and Lal et al (2002,  Drug Discov Today  15;7(18 Suppl):S143-9). 
     Typically the array is a microarray. By “microarray” we include the meaning of an array of regions having a density of discrete regions of at least about 100/cm 2 , and preferably at least about 1000/cm 2 . The regions in a microarray have typical dimensions, e.g. diameter, in the range of between about 10-250 μm, and are separated from other regions in the array by about the same distance. The array may alternatively be a macroarray or a nanoarray. 
     Once suitable binding molecules (discussed above) have been identified and isolated, the skilled person can manufacture an array using methods well known in the art of molecular biology; see Examples below. 
     A third aspect of the present invention provides the use of one or more (preferably two or more) biomarkers selected from the group defined in Table 3A and/or Table 3B in combination for identifying hypersensitivity response sensitising agents. Preferably, all of the biomarkers defined in Table 3A and Table 3B are used collectively for identifying hypersensitivity response sensitising agents. Preferably, the use is consistent with the method described in the first aspect of the invention, and the embodiments described therein. 
     A fourth aspect of the invention provides an analytical kit for use in a method according the first aspect of the invention, comprising or consisting of:
         A) an array according to the second aspect of the invention; and   B) instructions for performing the method according to the first aspect of the invention (optional).       

     The analytical kit may comprise one or more control agents. Preferably, the analytical kit comprises or consists of the above features, together with one or more negative control agents and/or one or more positive control agents. 
    
    
     
       Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures: 
         FIG. 1 . Phenotype of MUTZ-3 cells prior to stimulation with sensitizing and non-sensitizing chemicals 
       Cell surface expression levels of CD14, CD1a, CD34, CD54, CD80, CD86 and HLA-DR were assessed with flow cytometry. Gates were set to exclude debris and dead cells, and quadrants were established by comparing with relevant isotype controls. Results are shown from one representative experiment out of six. 
         FIGS. 2A and 2B . Changes in CD86 expression following stimulation with sensitizing and non-sensitizing chemicals 
       Cell surface expression levels of CD86 were monitored after stimulation with chemicals for 24 h.  FIG. 2A ) Chemical-induced upregulation of CD86, in terms of changes in frequency of positive cells, were determined by flow cytometry, as exemplified by the comparison of 2-aminophenol-stimulated cells (right dotplot) and unstimulated controls (left dotplot). Results are shown from one representative experiment out of three. Gates were set to exclude debris and dead cells, and quadrants were established by comparing with relevant isotype controls.  FIG. 2B ) Compilation of frequencies of CD86-positive cells after 24 h of stimulation. Statistical analysis was performed using paired Student&#39;s t test. *p&lt;0.05, #p&lt;0.01, NA, not analysed. 
         FIGS. 3A-3D . Principal component analysis of transcripts differentially expressed after chemical stimulation 
       mRNA levels in MUTZ-3 cells stimulated for 24 h with 20 sensitizing and 20 non-sensitizing chemicals were assessed with transcritomics using Affymetrix Human Gene 1.0 ST arrays. Structures and similarities in the gene expression dataset were investigated using principal component analysis (PCA) in the software Qlucore.  FIG. 3A ) PCA of genes differentially expressed in cells stimulated with sensitizing (red) as compared to non-sensitizing (green) chemicals (1010 genes identified with one-way ANOVA).  FIG. 3B ) PCA of genes differentially expressed in cells stimulated with sensitizing as compared to non-sensitizing chemicals (1010 genes), but now samples are coloured by the compound used for stimulation.  FIG. 3C ) PCA of genes differentially expressed when comparing the different stimulations with 2-way ANOVA (1137 genes). Samples are coloured according to sensitizing (red) and non-sensitizing (green) chemicals.  FIG. 3D ) PCA of genes differentially expressed when comparing the different stimulations with 2-way ANOVA (1137 genes), but now samples are coloured by the compound used for stimulation. P, p-value from ANOVA. Q, p-value corrected for multiple hypothesis testing. 
         FIGS. 4A-4C . Identification and PCA analysis of “Prediction Signature” 
         FIG. 4A ) The number of differentially expressed significant genes in cells stimulated with sensitizing as compared to non-sensitizing chemicals (1010 genes) was reduced using Backward Elimination. The lowest KLD is observed after elimination of 810 analytes, referred to as the Breakpoint. The remaining 200 genes is considered to be the top predictors in the data set, and is termed “Prediction Signature”.  FIG. 4B ) Complete separation between sensitizers (red) and non-sensitizers (green) is observed with PCA of the “Prediction Signature”.  FIG. 4C ) Same PCA as in B, now with samples coloured according to their potency in LLNA. 
         FIG. 5 . Validation of selection procedure of “Prediction Signature” 
       The method by which the “Prediction Signature” was constructed was validated by repeating the process on 70% of the data set, selected at random. The remaining 30% of data was used as a test set for signature validation. A) PCA demonstrates that the “Test Gene Signature” can separate sensitizers from non-sensitizers. Only the samples of the 70% training set, displayed in bright colours, were used to build the space of the first three principal components. The test set samples, displayed in dark colours, were plotted into this space based on expression levels of the analytes in the “Test Gene Signature”. B) An SVM was trained on the 70% training set, and validated with the 30% test set. The area under the ROC curve of 1.0 proves that the group belonging of all samples in the test set was correctly predicted, demonstrating the strength of the “Test Gene Signature”, and by association also the strength of our “Prediction Signature”. 
         FIG. 6 . The interactome of the “Prediction Signature” 
       Interactome of the 200 molecules (orange) and molecules connecting theses according to evidence from IPA. Direct interactions are shown as solid lines and indirect as dotted lines. 
     
    
    
     EXAMPLES 
     Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This is commonly caused by immunological responses towards chemical haptens leading to substantial economic burden for society. 
     Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within e.g. pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. 
     We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation with 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an accuracy of 100%, sensitivity of 100% and specificity of 100%. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. 
     A gene signature predicting sensitization using a human cell line in vitro has been developed. This easy and robust cell-based assay can completely replace or drastically reduce current test systems, using experimental animals. Being based on human biology, the assay is considered to be more relevant and more accurate for predicting sensitization in humans than the traditional animal-based tests. 
     Results 
     The Cellular Rational of the In Vitro Cell Culture System 
     Acting as the link between innate and adaptive immunity, DCs are essential immunoregulatory cells of the immune system. Their unique property to recognize antigen for the purpose of initiating T cell responses, and their potent regulatory function in skewing immune responses, makes them targets for assay development. However, primary DCs constitute a heterogeneous and minor population of cells not suited for screening. The obvious advantages of using a cell line with characteristics compared to primary DCs for the basis of a predictive test are stability, reproducibility and unlimited supply of cells. So far, no leukemia with obvious DC-like properties has been reported, probably due to the fact that the characteristics of this cell type are determined by a complex terminal differentiation process that can occur only post-cell division [11], and thus, the generation of DC-like cell lines relies on available myeloid leukemia cell lines. MUTZ-3 is a human acute myelomonocytic leukemia cell line with a potent ability to differentiate into DCs, present antigens and induce specific T-cell proliferation. Among the available myeloid human cell lines, MUTZ-3 is by far the preferred candidate. Similar to immature primary DCs, MUTZ-3 progenitor express CD1a, HLA-DR and CD54, as well as low levels of CD80 and CD86 ( FIG. 1 ). The MUTZ-3 population also contains three subpopulations of CD14 + , CD34 +  and double negative cells, previously reported to be transitional differentiation steps from a proliferative CD34 +  progenitor into a non-proliferative CD14 +  DC precursor [9]. Consequently, we utilized constitutively differentiating progenitor MUTZ-3 cells as the basis for the test system. 
     CD86 surface expression in response to sensitizer stimulation: CD86 is the most extensively studied biomarker for sensitization to date, in cell-systems such as monocyte derived dendritic cells (MoDCs) or dendritic cell-like human cell lines and their progenitors, such as THP-1, U-937 and KG-1. Thus, as a reference, cell surface expression of CD86 was measured with flow cytometry after 24 h stimulation with 20 sensitizers and 20 non-sensitizers, as well as with vehicle controls (Table 1). CD86 was significantly up-regulated on cells stimulated with 2-Aminophenol, Kathon CG, 2-nitro-1,4-Phenylendiamine, 2,4-Dinitrochlorobenzene, 2-Hydroxyethyl acrylate, Cinnamic aldehyde, p-Phenylendiamine, Resorcinol, and 2-Mercaptobenzothiazole. Hence, an assay based on measurement of a single biomarker, such as CD86, would give a sensitivity of 47% and a specificity of 100%. Consequently, CD86 cannot classify skin sensitizers, using a cell based system such as MUTZ-3. 
     Analysis of the transcriptional profiles in chemically stimulated MUTZ-3 cells: The genomic expression arrays were used to test 20 sensitizers and 20 non-sensitizers, in triplicates, and vehicle controls such as DMSO and distilled water, the latter in twelve replicates. In total, a data set was generated based on 144 samples. RMA normalization and quality controls of the samples revealed that the Oxazolone and Cinnamic aldehyde samples were significant outliers and had to be removed, or they would have dominated the data set prohibiting biomarker identification (data not shown). In addition, one of the replicates of potassium permanganate had to be removed due to a faulty array. This left a data set consisting of 137 samples, each with data from measurements of 33,297 transcripts. In order to mine the data set for information specific for sensitizers vs. non-sensitizers, the software Qlucore Omics Explorer 2.1 was used, which enable real time principal component analysis (PCA) analysis, while sorting the input genes after desired criteria, e.g. sensitizers and non-sensitizers, based on ANOVA p-value selection.  FIGS. 3A and 3B  show PCAs based on 1010 transcripts with a p-value of ≤2.0×10 −6  from ANOVA analysis, comparing sensitizing vs. non-sensitizing chemicals. As can be seen in  FIG. 3A , a clear distinction can be made between the two groups, with non-sensitizers forming a condensed cloud in the lower part of the figure (green), while sensitizers stretch upwards in various directions (red). However, a complete separation is not achieved between the two groups at this level of significance. From  FIG. 3B , now coloured according to stimulation, it is evident that one or more replicate of Glyoxal, Eugenol, Hexylcinnamic aldehyde, Isoeugenol, Resorcinol, Penicillin G and Ethylendiamine group together with the control group. In addition, one replicate or more of the non-sensitizers Tween 80, Octanoic acid and Phenol tend to group with the sensitizers.  FIGS. 3C and 3D  show PCA plots based on 1137 genes that all have a p-value of ≤7.0×10 −21 , when comparing the different stimulations. Identifying this large number of genes at this level of significance provides strong indications of the power of the data set. In  FIG. 3D , it is clear that the replicates group together, indicating high quality data. The triplicate samples of Potassium dichromate have a discrete profile, which demonstrate a substantial impact of the cells compared to non-sensitizers. 
     Furthermore, 2-Hydroxyethyl acrylate, 2-Aminophenol, Kathon CG, Formaldehyde, 2-nitro-1,4-Phenylendiamine, 2,4-Dinitrochlorobenzoic acid, p-Phenylendiamine, 2-Mercaptobenzothiazole, Cinnamic alcohol and Resorcinol have replicates that group together, separate from the negative group. Still, as can be seen in  FIG. 3C  as well as in  3 A, complete separation is not achieved with neither of the gene signatures of 1010 and 1137 genes respectively. 
     Backward elimination identifies genes with the most discriminatory power: Even though the data set contains genes with p-values down to 1×10 −17 , lowering the p-value cutoff did not achieve complete separation between sensitizers and non-sensitizers. Gene signatures entirely selected on p-values does not provide the best possible predictive power, since a low p-value is no guarantee that a gene provides any additional information. To further reduce the number of transcripts for a predictive biomarker signature, we employed an algorithm for backward elimination ( FIG. 4A ). The algorithm removes genes one by one while taking into account not only the impact of genes individually, but how they perform collectively with the entire selected gene signature. For each gene eliminated, the Kullback-Leibler divergence (KLD) value is lowered, until a breakpoint is reached, at which point 200 genes remained. Continuing eliminating genes at this point causes the KLD to rise again, indicating that information is being lost ( FIG. 4A ). Therefore, the 200 genes with lowest KLD value were selected for further analysis. PCA of the 200 analytes revealed that they have the ability to completely separate sensitizers vs. non-sensitizers, indicating that these transcripts can be used as predictors for sensitizing properties of unknown samples ( FIG. 4B ). Importantly, by coloring the samples in the PCA by their potency, according to LLNA, it is clear that potency can be predicted by these genes, as well ( FIG. 4C ). The 200 genes are termed the “Prediction Signature” and their identity is listed in Table 3. 
     Validation of the analysis method used to identify the Prediction Signature: To validate the predictive power of our signature, we used a supervised learning method called the Support Vector Machine (SVM) [12], which maps the data from a training set in space in order to maximize the separation of gene expression induced by sensitizing and non-sensitizing chemicals. As training set, 70% of the data set was selected randomly and the entire selection process (as described above) was repeated. Starting with 29,141 transcripts, the signature was reduced to 200 transcripts, termed “Test Gene Signature”, using ANOVA filtering and backward elimination. The remaining 30% of the data set was used to test the signature obtained. The partitioning of the data set into subsets of 70% training data set and 30% test data set was done in a stratified random manner, meaning that the proportion of sensitizers and non-sensitizers in the complete data set are maintained in both the subsets, although the samples included in either of the two subsets are selected at random. Thereafter, the “Test Gene Signature” was used to train an SVM model with the training set, and the predictive power of the model was assessed with the test set.  FIG. 5A  shows a PCA plot based on the “Test Gene Signature” and the samples of the test set. Clearly, the separation between sensitizers (red) and non-sensitizers (green) resembles the one observed for the “Prediction Signature” in  FIG. 4B . In the PCA of  FIG. 5A , the samples of the sensitizing and non-sensitizing chemicals of the test set have been colored dark red and dark green respectively, indicating that they are not contributing to the principal components of the plot, but are merely plotted based on their expression values of the selected “Test Gene Signature”. As can be seen, sensitizers from the test set group with sensitizers from the training set, while non-sensitizers from the test set group with non-sensitizers from the training set. This is a very intuitive way of predicting the group belonging of the samples in the test set, using only the pattern recognition of the eye. The outcome of the SVM training and validation can be seen in  FIG. 5B , where an area under the ROC curve of 1 confirms the ability of the “Test Gene Signature” to predict which group the sample belonged to in the test set. The prediction performance of the assay reveals an accuracy of 96%, sensitivity of 100% and specificity of 92%. While this experiment does not validate the prediction power of the “Prediction Signature” per se, it does indeed validate the method by which it has been selected, supporting the claim that the “Prediction Signature” is capable of accurately predict sensitizing properties of unknown samples. 
     Molecular functions and canonical pathways of the “Prediction Signature”. 
     Using Ingenuity Pathways Analysis (IPA, Ingenuity Systems Inc.), 184 of the 200 molecules in the signature were characterized with regard to functions and known (canonical) pathways. The remaining 16 molecules could not be mapped to any IPA entries. The dominating functions identified were small molecule biochemistry (38 molecules), cell death (33), lipid metabolism (24), hematological system development (19), cellular growth and proliferation (16), molecular transport (15), cell cycle (15) and carbohydrate metabolism (15), see table 4 for details. 67 of the 184 molecules were involved in the listed functions. Of the remaining 117 molecules, 30 were known from a variety of human diseases and molecular functions, such as described biomarkers (SCARB2, RFC2, VPS37A and BCL7A) and drug targets (ABAT). Most of these molecules are metabolic markers. In the signature as a whole, there are several drug targets, such as HMGCR, HMOX1, ABAT, RXRA, CD33, MAP2K1, MAPK13 and CD86. Two are described for skin disorders: CD86 (psoriasis) and RXRA (eczema). The signature also contains skin development (DHCR24) and dendritic cell markers (MAP2K1, NLRP12 and RFC2). 
     Pathways possibly invoked by the molecules in the signature were also investigated using IPA. Those most highly populated were NRF2 mediated oxidative response (10), xenobiotic metabolism signaling (8), LPS/IL-1 mediated inhibition of RXR function (6), aryl hydrocarbon receptor signaling (6) and protein kinase A signaling (6). The five highest ranked of these pathways are all known to take part in reactions provoked by foreign substances, xenobiotics. Xenobiotics are natural or synthetic chemical compounds, foreign to the human body. 
     Conclusions 
     Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by dysregulated adaptive immune responses to allergens [13]. Small molecular weight chemicals, so-called haptens, can bind self-proteins in the skin, which enables internalisation of the protein-bound allergenic chemical by skin dendritic cell (DC). DCs, under the influence of the local microenvironment, process the protein-hapten complex, migrate to the local lymph nodes and activate naive T cells. The initiation and development of allergen-specific responses, mainly effector CD8+ T cells and Th1 cells, and production of immunoregulatory proteins, are hallmarks of the immune activation observed in ACD. This T-cell mediated type IV hypersensitivity reaction is characterised by symptoms such as rash, blisters and itching. ACD is the most common manifestation of immunotoxicity observed in humans [13] and hundreds of chemicals have been shown to cause sensitization in skin [14]. The driving factors and molecular mechanisms involved in sensitization are still unknown even though intense research efforts have been carried out to identify the immunological responses towards allergenic chemicals. The REACH legislation requires that all chemicals produced over 1 ton/year are tested for hazardous properties such as toxicity and allergenicity [5], which increase the demand for accurate assays for predictive power. Today, the identification of potential human sensitizers relies on animal experimentation, in particular the murine local lymph node assay (LLNA) [6]. The LLNA is based upon measurements of proliferation induced in draining lymph nodes of mice after chemical exposure [15]. Chemicals are defined as sensitizers if they provoke a three-fold increase in proliferation compared to control, and the amount of chemical required for the increase is the EC3 value. Thus, the LLNA can also be used to categorize the chemicals based on sensitisation potency. However, LLNA is in many ways not optimal. Besides the obvious ethical reasons, the assay is also time consuming and expensive. Human sensitization data often stem from human maximization tests (HMT) [16] and human patch tests (HPT). In an extensive report from the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the performance characteristics of LLNA were compared to other available animal-based methods and human sensitization data (HMT and HPT) [17]. The LLNA performance in comparison to human data (74 assessments) revealed an accuracy of 72%, a sensitivity of 72% and a specificity of 67%. Accuracy is defined as the proportion of correct outcomes of a method, sensitivity is the proportion of all positive chemicals that are correctly classified as positives, and specificity is the proportion of all negative chemicals that are correctly classified as negatives. Thus, there is a clear need to develop more reliable test methods for sensitization. Additionally, the 7th Amendment to the Cosmetics Directive (76/768/EEC) poses a complete ban on using animal experimentation for testing cosmetic ingredients by 2013 when a scientific reliable method is available. Thus, there is a great need from the industry for reliable predictive test methods that are based on human cells. Various human cell lines and primary cells involved in sensitization have been evaluated as predictive test system, such as epithelial cells, dendritic cells and T cells, however no validated test assay is currently available. Various single biomarkers have been suggested to be upregulated upon stimulation with sensitizing chemicals, such as CD40, CD80, CD54, CXCL8, IL-1β, MIP-1β, p38 MAPK as reviewed in [18], yet singlehanded, none of them have the predictive power to discriminate between sensitizing and non-sensitizing chemicals. CD86 is among the markers most extensively studied; however, determining the expression level of this marker in our assay is relevant but not sufficient as readout for sensitization ( FIG. 2 ). Only 9 out of 19 sensitizing chemicals induced a significant upregulation of CD86. Instead, it is our firm belief that the analyses of biomarker signatures are superior to any single biomarker. We therefore utilized the power of complete-genome transcriptomics and screened the gene regulation induced by a large set of well-defined chemicals and controls. The large number of differentially expressed genes in MUTZ-3 cells stimulated with sensitizing chemicals as compared to non-sensitizing controls, as identified with ANOVA ( FIG. 3 ) revealed that MUTZ-3 indeed has a capacity to differentiate between these two groups, thus being a suitable cell system for in vitro assays for sensitization. Efforts have been made to create assays based on full genome analysis in various cell systems, such as CD34 + -progenitor cells-derived DCs [19-21]. While such assays might provide in vivo like environments, one could argue that primary cells are not well suited for an assay of this great commercial interest. Moreover, the ethical aspect needs to be considered in such a system. Furthermore, previous efforts within in vitro assay development for sensitization that rely on full genome analysis have used a very limited set of testing compounds. To date, this study is the largest study performed within this area, utilizing 20 positive and 20 negative training compounds. Efforts have been made by us to divide these training compounds into two subsets, for training and testing respectively. While these experiments have resulted in successful predictions (data not shown), it is our experience that sensitizing compounds differ greatly in their induced gene expression profile, as can be seen in  FIG. 3D . In this perspective, we wished to include as many training compounds as possible when identifying our “Prediction Signature”. Thus, we did not exclude any compounds for validation. 
     Instead, we have validated the method by which the “Prediction Signature” was identified, by subdividing the samples into training and test sets at random, using unseen data for validation, as seen in  FIG. 5 . By including all sensitizing compounds, with a wide range of reactive mechanisms as well as sensitizing potencies, we argue that we have identified a prediction signature that is well suited for predicting sensitizing properties of unknown samples. Of important note, our “Prediction Signature” is able to predict the potency of sensitizing compounds, such as those defined by the LLNA, as is demonstrated in  FIG. 4C . However, the potency predicted by LLNA and that of our classifier do not match for all samples. Notably, the moderate sensitizer 2-Hydroxyethyl acrylate show strong resemblance of strong and extreme sensitizers with respect to their gene expression profile. Similarly, the moderate sensitizers Ethylendiamine, Hexylcinnamic aldehyde, and Glyoxal group together with weak sensitizers. These findings provide vast implications that sensitizing potency, as defined, may need revising. We argue that our “Prediction Signature” may be used for such classifications. 
     In conclusion, we present an in vitro assay, based on a MUTZ-3 cell system that with an identified “Prediction Signature” consisting of 200 genes, which have the ability to correctly classify a sample as sensitizer or non-sensitizer. In addition, this assay can predict the potency of sensitizing compounds, and may be used to revise such classifications. 
     Materials and Methods 
     Chemicals 
     A panel of chemicals consisting of 20 sensitizers and 20 non-sensitizers were used for cell stimulations. The sensitizers were 2,4-Dinitrochlorobenzene, Cinnamaldehyde, Resorcinol, Oxazolone, Glyoxal, 2-Mercaptobenzothiazole, Eugenol, Isoeugenol, Cinnamic alcohol and p-Phenylendiamine, Formaldehyde, Ethylendiamine, 2-Hydroxyethyl acrylate, Hexylcinnamic aldehyde, Potassium Dichromate, Penicillin G, Katchon CG (MCI/MI), 2-aminophenol, Geraniol and 2-nitro-1,4-Phenylendiamine (Table 1). The non-sensitizers were Sodium dodecyl sulphate, Salicylic acid, Phenol, Glycerol, Lactic acid, Chlorobenzene, p-Hydrobenzoic acid, Benzaldehyde, Diethyl Phtalate and Octanoic acid, Zinc sulphate, 4-Aminobenzoic acid, Methyl salicylate, Ethyl vanillin, Isopropanol, Dimethyl formamide, 1-Butanol, Potassium permanganate, Propylene glycol and Tween 80 (Table 1). All chemicals were from Sigma-Aldrich, St. Louis, Mo., USA. Compounds were dissolved in either Dimethyl sulfoxide (DMSO) or distilled water. Prior to stimulations, the cytotoxicity of all compounds were monitored, using Propidium Iodide (PI) (BD Biosciences, San Diego, Calif.) using protocol provided by the manufacturer. The relative viability of stimulated cells was calculated as 
     
       
         
           
             
               Relative 
                
               
                   
               
                
               viability 
             
             = 
             
               
                 
                   fraction 
                    
                   
                       
                   
                    
                   of 
                    
                   
                       
                   
                    
                   viable 
                    
                   
                       
                   
                    
                   stimulated 
                    
                   
                       
                   
                    
                   cells 
                 
                 
                   fraction 
                    
                   
                       
                   
                    
                   of 
                    
                   
                       
                   
                    
                   viable 
                    
                   
                       
                   
                    
                   unstimulated 
                    
                   
                       
                   
                    
                   cells 
                 
               
               · 
               100 
             
           
         
       
     
     For toxic compounds, the concentration yielding 90% relative viability (Rv90) was used. For untoxic compounds, a concentration of 500 μM was used when possible. For non-toxic compounds that were insoluble at 500 μM in medium, the highest soluble concentration was used. For compounds dissolved in DMSO, the in-well concentration was 0.1% DMSO. The vehicle and concentrations used for each compound are listed in Table 2. 
     Chemical Exposure of the Cells 
     The human myeloid leukaemia-derived cell line MUTZ-3 (DSMZ, Braunschweig, Germany) was maintained in a-MEM (Thermo Scientific Hyclone, Logan, Utah) supplemented with 20% (volume/volume) fetal calf serum (Invitrogen, Carlsbad, Calif.) and 40 ng/ml rhGM-CSF (Bayer HealthCare Pharmaceuticals, Seattle, Wash.), as described [10]. Prior to each experiment, the cells were immunophenotyped using flow cytometry as a quality control. Cells were seeded in 6-well plates at 200.000 cells/ml. Stock solutions of each compound was prepared in either DMSO or distilled water, and were subsequently diluted so the in-well concentrations corresponded to the Rv90 value, and in-well concentrations of DMSO were 0.1%. Cells were incubated for 24 h at 37° C. and 5% CO 2 . Thereafter, cells were harvested and analysed with flow cytometry. In parallel, harvested cells were lysed in TRIzol reagent (Invitrogen) and stored at −20° C. until RNA extraction. Stimulations with chemicals were performed in three individual experiments, so that triplicates samples were obtained. 
     Phenotypic Analysis with Flow Cytometry 
     All cell surface staining and washing steps was performed in PBS containing 1% BSA (w/v). Cells were incubated with specific mouse mAbs for 15 min at 4° C. The following mAbs were used for flow cytometry: FITC-conjugated CD1a (DakoCytomation, Glostrup, Denmark), CD34, CD86, and HLA-DR (BD Biosciences), PE-conjugated CD14 (DakoCytomation), CD54 and CD80 (BD Biosciences). Mouse IgG1, conjugated to FITC or PE were used as isotype controls and PI was used to assess cell viability (BD Biosciences). FACSDiva software was used for data acquisition with FACSCanto II instrument (BD Bioscience). 10,000 events were acquired and gates were set based on light scatter properties to exclude debris and nonviable cells. Further data analysis was performed using FCS Express V3 (De Novo Software, Los Angeles, Calif.). 
     Preparation of cRNA and Gene Chip Hybridization 
     RNA isolation and gene chip hybridization was performed as described [22]. Briefly, RNA from unstimulated and chemical-stimulated MUTZ-3 cells, from triplicate experiments, were extracted and analysed. The preparation of labeled sense DNA was performed according to Affymetrix GeneChip Whole Transcript (WT) Sense Target Labeling Assay (100 ng Total RNA Labeling Protocol) using the recommended kits and controls (Affymetrix, Santa Clara, Calif.). Hybridization, washing and scanning of the Human Gene 1.0 ST Arrays were performed according to the manufacturer&#39;s protocol (Affymetrix). 
     Microarray Data Analysis and Statistical Methods 
     The microarray data were normalised and quality checked with the RMA algorithm, using Affymetrix Expression Console (Affymetrix). Genes that were significantly regulated when comparing sensitizers with non-sensitizers were identified using one-way ANOVA, with false discovery rate (FDR) as a correction for multiple hypothesis testing. In order to reduce the large number of identified significant gene, we applied an algorithm developed in-house for Backward Elimination of analytes (Carlsson et al, unpublished). With this method, we train and test a Support Vector Machine (SVM) model [12] with leave-one out cross-validation, with one analyte left out. This process is iterated until each analyte has been left out once. For each iteration, a Kullback-Leibler divergence (KLD) is recorded, yielding N KLDs, where N is the number of analytes. The analyte that was left out when the smallest KLD was observed is considered to provide the least information in the data set. Thus, this analyte is eliminated and the iterations proceed, this time with N-1 analytes. In this manner, the analytes are eliminated one by one until a panel of markers remain that have been selected based on each analyte&#39;s ability to discriminate between sensitizers and non-sensitizers. The script for Backwards Eliminations was programmed for R [23], with the additional package e1071 [24]. ANOVA analyses and visualisation of results were performed in Qlucore Omics Explorer 2.1 (Qlucore, Lund, Sweden). The selected biomarker profile of 200 transcripts were designated the “Prediction Signature”. 
     Validation of the method for identification of the “Prediction Signature” In the absence of an external test data set, the data set was divided into a training set of 70% and a test set of 30% of the samples. The division was performed randomly, while maintaining the proportions of sensitizers and non-sensitizers in each subset at the same ratio as in the complete data set. A biomarker signature was identified in the training set using ANOVA filtering and Backward Elimination, as described above. This test signature was used to train an SVM, using the training set, which was thereafter applied to predict the samples of the test set. The distribution of the area under the Receiver Operating Characteristic (ROC) curve [25] was used as a measurement of the performance of the model. 
     Assessment of Biological Functions of “Prediction Signature” Using Pathway Analysis 
     In order to investigate the biological functions the gene profile was analyzed using the Ingenuity Pathway Analysis software, IPA, (Ingenuity Systems, Inc. Mountain View, USA). The 200 top genes resulting from Backward Elimination were analyzed using the ‘build’ and ‘Path Explorer’ functions to build an interactome of the core genes from the “Prediction Signature” and connecting molecules suggested by IPA. The 200 molecules in the “Prediction Signature” were connected using the shortest known paths. In this process only human evidence from primary cells, cell lines and epidermal tissue was used. All molecules except for endogenous and chemical drugs were allowed in the network and all kinds of connections were allowed. Known ‘Functions’ and ‘Canonical Pathways’ from IPA were mapped to the interactome using the ‘Overlay’ function. 
     Abbreviations 
     ACD, atopic contact dermatitis; 
     AML, acute myeloid leukemia cell; 
     APC, Antigen Presenting Cell; 
     DC, Dendritic Cell; 
     GM-CSF, Granulocyte macrophage colony-stimulating factor; 
     GPMT, Guinea pig maximization test; 
     HMT, Human Maximation Test; 
     HPTA, Human Patch Test Allergen; 
     IL, Interleukin; 
     LLNA, Local Lymph Node Assay; 
     PCA, Principal Component Analysis 
     REFERENCES 
     
         
         1. Akhavan, A. and S. R. Cohen,  The relationship between atopic dermatitis and contact dermatitis.  Clin Dermatol, 2003. 21(2): p. 158-62. 
         2. Mortz, C. G., et al.,  Prevalence of atopic dermatitis, asthma, allergic rhinitis, and hand and contact dermatitis in adolescents. The Odense Adolescence Cohort Study on Atopic Diseases and Dermatitis.  Br J Dermatol, 2001. 144(3): p. 523-32. 
         3. Nielsen, N. H., et al.,  Allergic contact sensitization in an adult Danish population: two cross - sectional surveys eight years apart  ( the Copenhagen Allergy Study ). Acta Derm Venereol, 2001. 81(1): p. 31-4. 
         4. Fonacier, L. S., S. C. Dreskin, and D. Y. Leung,  Allergic skin diseases.  J Allergy Clin Immunol. 125(2 Suppl 2): p. S138-49. 
         5.  EC  1907/2006, in  Regulation  ( EC )  No  1907/2006  of the European Parliament and of the Council of  18  Dec.  2006  concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals  ( REACH ),  establishing a European Chemicals Agency, amending Directive  1999/45/ EC and repealing Council Regulation  ( EEC )  No  793/93  and Commission Regulation  ( EC )  No  1488/94  as well as Council Directive  76/769/ EEC and Commission Directives  91/155/ EEC,  93/67/ EEC,  93/105/ EC and  2000/21/ EC.    
         6. Basketter, D. A., et al.,  Local lymph node assay—validation, conduct and use in practice.  Food Chem Toxicol, 2002. 40(5): p. 593-8. 
         7. Magnusson, B. and A. M. Kligman,  The identification of contact allergens by animal assay. The guinea pig maximization test.  J Invest Dermatol, 1969. 52(3): p. 268-76. 
         8. Masterson, A. J., et al.,  MUTZ -3,  a human cell line model for the cytokine - induced differentiation of dendritic cells from CD 34 + precursors.  Blood, 2002. 100(2): p. 701-3. 
         9. Santegoets, S. J., et al.,  A CD 34(+)  human cell line model of myeloid dendritic cell differentiation: evidence for a CD 14(+) CD 11 b (+)  Langerhans cell precursor.  J Leukoc Biol, 2006. 80(6): p. 1337-44. 
         10. Larsson, K., M. Lindstedt, and C. A. Borrebaeck,  Functional and transcriptional profiling of MUTZ -3,  a myeloid cell line acting as a model for dendritic cells.  Immunology, 2006. 117(2): p. 156-66. 
         11. Rasaiyaah, J., et al.,  Dendritic cells and myeloid leukaemias: plasticity and commitment in cell differentiation.  Br J Haematol, 2007. 138(3): p. 281-90. 
         12. Cortes, C. and V. Vapnik,  Support - Vector Networks.  Machine Learning, 1995. 20(3): p. 273-297. 
         13. Kimber, I., et al.,  Allergic contact dermatitis.  Int Immunopharmacol, 2002. 2(2-3): p. 201-11. 
         14. Gerberick, G. F., et al.,  Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods.  Dermatitis, 2005. 16(4): p. 157-202. 
         15. Kimber, I., et al.,  Classification of contact allergens according to potency: proposals.  Food Chem Toxicol, 2003. 41(12): p. 1799-809. 
         16. Kligman, A. M.,  The identification of contact allergens by human assay.  3.  The maximization test: a procedure for screening and rating contact sensitizers.  J Invest Dermatol, 1966. 47(5): p. 393-409. 
         17. Haneke, K. E., et al.,  ICCVAM evaluation of the murine local lymph node assay. Data analyses completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods.  Regul Toxicol Pharmacol, 2001. 34(3): p. 274-86. 
         18. dos Santos, G. G., et al.,  Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound.  Toxicol Appl Pharmacol, 2009. 236(3): p. 372-82. 
         19. Hooyberghs, J., et al.,  A cell - based in vitro alternative to identify skin sensitizers by gene expression.  Toxicol Appl Pharmacol, 2008. 231(1): p. 103-11. 
         20. Lambrechts, N., et al.,  Gene markers in dendritic cells unravel pieces of the skin sensitization puzzle.  Toxicol Lett, 2010. 196(2): p. 95-103. 
         21. Lambrechts, N., et al.,  Assessment of chemical skin - sensitizing potency by an in vitro assay based on human dendritic cells.  Toxicol Sci, 2010. 116(1): p. 122-9. 
         22. Lindstedt, M., et al.,  Genomic and functional delineation of dendritic cells and memory T cells derived from grass pollen - allergic patients and healthy individuals.  Int Immunol, 2005. 17(4): p. 401-9. 
         23. R Development Core Team.  R: A language and environment for statistical computing. R Foundation for Statistical Computing.  2008; Available from: www.R-project.org. 
         24.  R package e 1071. Available from: cran.r-project.org/web/packages/e1071/index.html. 
         25. Lasko, T. A., et al.,  The use of receiver operating characteristic curves in biomedical informatics.  J Biomed Inform, 2005. 38(5): p. 404-15. 
         26. Warbrick, E. V., et al.,  Influence of application vehicle on skin sensitization to methylchloroisothiazolinone/methylisothiazolinone: an analysis using the local lymph node assay.  Contact Dermatitis, 1999. 41(6): p. 325-9. 
         27. Roberts, D. W., et al.,  Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitization.  Chem Res Toxicol, 2007. 20(7): p. 1019-30. 
         28. Gerberick, G. F., et al.,  Contact allergenic potency: correlation of human and local lymph node assay data.  Am J Contact Dermat, 2001. 12(3): p. 156-61. 
         29. Basketter, D. A., D. Sanders, and I. R. Jowsey,  The skin sensitization potential of resorcinol: experience with the local lymph node assay.  Contact Dermatitis, 2007. 56(4): p. 196-200. 
         30. Ryan, C. A., et al.,  Activity of human contact allergens in the murine local lymph node assay.  Contact Dermatitis, 2000. 43(2): p. 95-102. 
         31.  APPENDIX C; Comparative LLNA: BrdU - FC, Traditional LLNA, Guinea Pig Skin Sensitization, and Human Data.  2008 Available from: iccvam.niehs.nih.gov/methods/immunotox/fcLLNA/Appx/AppendixC_LLNA_F C07Jan08FD.pdf. 
         32. Patlewicz, G., et al.,  Skin - sensitization structure - activity relationships for aldehydes.  Contact Dermatitis, 2001. 44(6): p. 331-6. 
         33. Basketter, D. A., G. F. Gerberick, and I. Kimber,  Strategies for identifying false positive responses in predictive skin sensitization tests.  Food Chem Toxicol, 1998. 36(4): p. 327-33. 
         34. Ashby, J., et al.,  Structure activity relationships in skin sensitization using the murine local lymph node assay.  Toxicology, 1995. 103(3): p. 177-94. 
         35. Sakaguchi, H., et al.,  Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test  ( h - CLAT ).  II. An inter - laboratory study of the h - CLAT.  Toxicol In Vitro, 2006. 20(5): p. 774-84. 
         36.  The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds.  1999; Available from: iccvam.niehs.nih.gov/docs/immunotox_docs/IIna/IInarep.pdf. 
       
    
     TABLES 
       
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of sensitizing and non-sensitizing chemicals, based on murine LLNA 
               
               
                 classification, tested in the cell-based assay. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Compound 
                 Abbreviation 
                 Potency 
                 LLNA 
                 HMT 1   
                 HPTA 1   
               
               
                   
               
               
                 Sensitizers 
                   
                   
                   
                   
                   
               
               
                 2,4-Dinitrochlorobenzene 
                 DNCB 
                 Extreme [15] 
                 +[15] 
                   
                   
               
               
                 Oxazolone 
                 OXA 
                 Extreme [15] 
                 +[15] 
                   
                   
               
               
                 Potassium dichromate 
                 PD 
                 Extreme [14] 
                 +[14] 
                 + 
                 + 
               
               
                 Kathon CH (MC/MCI) 
                 KCG 
                 Extreme [14, 26] 
                 +[14, 26] 
                   
                   
               
               
                 Formaldehyde 
                 FA 
                 Strong [15] 
                 +[15] 
                 + 
                 + 
               
               
                 2-Aminophenol 
                 2AP 
                 Strong [27] 
                 +[27] 
                   
                   
               
               
                 2-nitro-1,4-Phenylendiamine 
                 NPDA 
                 Strong [27] 
                 +[27] 
                   
                   
               
               
                 p-Phenylendiamine 
                 PPD 
                 Strong [28] 
                 +[28] 
                 + 
                 + 
               
               
                 Hexylcinnamic aldehyde 
                 HCA 
                 Moderate [15] 
                 +[15] 
                   
                   
               
               
                 2-Hydroxyethyl acrylate 
                 2HA 
                 Moderate [27] 
                 +[27] 
                 + 
                   
               
               
                 2-Mercaptobenzothiazole 
                 MBT 
                 Moderate [27] 
                 +[27] 
                 + 
                 + 
               
               
                 Glyoxal 
                 GO 
                 Moderate [27] 
                 +[27] 
                 + 
                   
               
               
                 Cinnamaldehyde 
                 CALD 
                 Moderate [28] 
                 +[28] 
                 + 
                 + 
               
               
                 Isoeugenol 
                 IEU 
                 Moderate [28] 
                 +[28+ 
                 + 
                   
               
               
                 Ethylendiamine 
                 EDA 
                 Moderate [14] 
                 +[14] 
                   
                   
               
               
                 Resorcinol 
                 RC 
                 Moderate [29] 
                 +[29] 
                 − 
                 + 
               
               
                 Cinnamic alcohol 
                 CALC 
                 Weak [27] 
                 +[28] 
                   
                   
               
               
                 Eugenol 
                 EU 
                 Weak [28] 
                 +[28] 
                   
                 + 
               
               
                 Penicillin G 
                 PEN G 
                 Weak [28] 
                 +[28] 
                 + 
                   
               
               
                 Geraniol 
                 GER 
                 Weak [14] 
                 +[14] 
                 − 
                 + 
               
               
                 Non-sensitizers 
                   
                   
                   
                   
                   
               
               
                 1-Butanol 
                 BUT 
                   
                 −[30] 
                   
                   
               
               
                 4-Aminobenzoic acid 
                 PABA 
                   
                 −[31] 
                 − 
                 + 
               
               
                 Benzaldehyde 
                 BA 
                   
                 −[32] 
                   
                   
               
               
                 Chlorobenzene 
                 CB 
                   
                 −[14] 
                   
                   
               
               
                 Diethyl phthalate 
                 DP 
                   
                 −[28] 
                   
                   
               
               
                 Dimethyl formamide 
                 OF 
                   
                 −[26] 
                   
                   
               
               
                 Ethyl vanillin 
                 EV 
                   
                 −[32] 
                   
                   
               
               
                 Glycerol 
                 GLY 
                   
                 −[28] 
                   
                   
               
               
                 Isopropanol 
                 IP 
                   
                 −[28] 
                   
                   
               
               
                 Lactic acid 
                 LA 
                   
                 −[14] 
                   
                   
               
               
                 Methyl salicylate 
                 MS 
                   
                 −[14] 
                 − 
                   
               
               
                 Octanoic acid 
                 OA 
                   
                 −[33] 
                   
                   
               
               
                 Propylene glycol 
                 PG 
                   
                 −[31] 
                   
                   
               
               
                 Phenol 
                 PHE 
                   
                 −[33] 
                 − 
                   
               
               
                 p-Hydroxybenzoic acid 
                 HBA 
                   
                 −[34] 
                   
                   
               
               
                 Potassium permanganate 
                 PP 
                   
                   
                   
                   
               
               
                 Salicylic acid 
                 SA 
                   
                 −[14] 
                 − 
                   
               
               
                 Sodium dodecyl sulphate 
                 SDS 
                   
                 + 2 [14, 33] 
                 − 
                   
               
               
                 Tween 80 
                 T80 
                   
                 −[35] 
                   
                 + 
               
               
                 Zinc sulphate 
                 ZS 
                   
                 + 2 [36] 
               
               
                   
               
               
                   1 HMT, Human Maximation Test; 
               
               
                 HPTA, Human Patch Test Allergen. Information is derived from [17]. 
               
               
                   2 False positives in LLNA. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Vehicle and concentrations used for testing. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Max 
                   
                 Concen- 
               
               
                   
                   
                   
                 solu- 
                   
                 tration 
               
               
                   
                 Abbre- 
                   
                 bility 
                 Rv90 
                 In culture  
               
               
                 Compound 
                 viation 
                 Vehicle 
                 (μM) 
                 (μM) 
                 (μM) 
               
               
                   
               
               
                 Sensitizers 
                   
                   
                   
                   
                   
               
               
                 2,4-Dinitrochlorobenzene 
                 DNCB 
                 DMSO 
                 — 
                 4 
                 4 
               
               
                 Oxazolone 
                 OXA 
                 DMSO 
                 250 
                 — 
                 250 
               
               
                 Potassium dichromate 
                 PD 
                 Water 
                 51.02 
                 1.5 
                 1.5 
               
               
                 Kathon CG (MC/MCI)* 
                 KCG 
                 Water 
                 — 
                 0.0035% 
                 0.0035% 
               
               
                 Formaldehyde 
                 FA 
                 Water 
                 — 
                 80 
                 80 
               
               
                 2-Aminophenol 
                 2AP 
                 DMSO 
                 — 
                 100 
                 100 
               
               
                 2-nitro-1,4- 
                 NPDA 
                 DMSO 
                 — 
                 300 
                 300 
               
               
                 Phenylendiamine 
                   
                   
                   
                   
                   
               
               
                 p-Phenylendiamine 
                 PPD 
                 DMSO 
                 566 
                 75 
                 75 
               
               
                 Hexylcinnamic aldehyde 
                 HCA 
                 DMSO 
                 32.34 
                 — 
                 32.24 
               
               
                 2-Hydroxyethyl acrylate 
                 2HA 
                 Water 
                 — 
                 100 
                 100 
               
               
                 2-Mercaptobenzothiazole 
                 MBT 
                 DMSO 
                 250 
                 — 
                 250 
               
               
                 Glyoxal 
                 GO 
                 Water 
                 — 
                 300 
                 300 
               
               
                 Cinnamaldehyde 
                 CALD 
                 Water 
                 — 
                 120 
                 120 
               
               
                 Isoeugenol 
                 IEU 
                 DMSO 
                 641 
                 300 
                 300 
               
               
                 Ethylendiamine 
                 EDA 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Resorcinol 
                 RC 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Cinnamic alcohol 
                 CALC 
                 DMSO 
                 500 
                 — 
                 500 
               
               
                 Eugenol 
                 EU 
                 DMSO 
                 649 
                 300 
                 300 
               
               
                 Penicillin G 
                 PEN G 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Geraniol 
                 GER 
                 DMSO 
                 — 
                 — 
                 500 
               
               
                 Non-sensitizers 
                   
                   
                   
                   
                   
               
               
                 1-Butanol 
                 BUT 
                 DMSO 
                 — 
                 — 
                 500 
               
               
                 4-Aminobenzoic acid 
                 PABA 
                 DMSO 
                 — 
                 — 
                 500 
               
               
                 Benzaldehyde 
                 BA 
                 DMSO 
                 250 
                 — 
                 250 
               
               
                 Chlorobenzene 
                 CB 
                 DMSO 
                 98 
                 — 
                 98 
               
               
                 Diethyl phthalate 
                 DP 
                 DMSO 
                 50 
                 — 
                 50 
               
               
                 Dimethyl formamide 
                 OF 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Ethyl vanillin 
                 EV 
                 DMSO 
                 — 
                 — 
                 500 
               
               
                 Glycerol 
                 GLY 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Isopropanol 
                 IP 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Lactic acid 
                 LA 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Methyl salicylate 
                 MS 
                 DMSO 
                 — 
                 — 
                 500 
               
               
                 Octanoic acid 
                 OA 
                 DMSO 
                 504 
                 — 
                 500 
               
               
                 Peopylene glycol 
                 PG 
                 Water 
                 — 
                 — 
                 500 
               
               
                 Phenol 
                 PHE 
                 Water 
                 — 
                 — 
                 500 
               
               
                 p-Hydroxybenzoic acid 
                 HBA 
                 DMSO 
                 250 
                 — 
                 250 
               
               
                 Potassium permanganate 
                 PP 
                 Water 
                 38 
                 — 
                 38 
               
               
                 Salicylic acid 
                 SA 
                 DMSO 
                 — 
                 — 
                 500 
               
               
                 Sodium dodecyl sulphate 
                 SDS 
                 Water 
                 — 
                 200 
                 200 
               
               
                 Tween 80 
                 T80 
                 DMSO 
                 — 
                 — 
                 500 
               
               
                 Zinc sulphate 
                 ZS 
                 Water 
                 126 
                 — 
                 126 
               
               
                   
               
               
                   1 Kathon CG is a mixture of the compounds MC and MCI. The concentration of this mixture is given in %. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Differentially expressed genes in MUTZ-3 cells stimulated with sensitizing 
               
               
                 chemicals as compared to non-sensitizing agents and controls. 
               
            
           
           
               
               
               
            
               
                   
                   
                 NCBI reference 
               
               
                 Gene Title 
                 Gene Symbol 
                 sequence 
               
               
                   
               
            
           
           
               
            
               
                 Table 3A 
               
            
           
           
               
               
               
            
               
                 fatty acid synthase 
                 FASN 
                 NM_004104 
               
               
                 squalene epoxidase 
                 SQLE 
                 NM_003129 
               
               
                 taste receptor, type 2, member 5 
                 TAS2R5 
                 NM_018980 
               
               
                 keratinocyte growth factor-like protein 1/2/hypothetical protein 
                 KGFLP1/2/ 
                 AF523265 
               
               
                 FLJ20444 
                 FLJ20444 
                   
               
               
                 transmembrane anterior posterior transformation 1 
                 TAPT1 
                 NM_153365 
               
               
                 Sprouty homolog 2 
                 SPRY2 
                 NM_005842 
               
               
                 B-cell CLL/lymphoma 7A 
                 BCL7A 
                 NM_020993 
               
               
                 solute carrier family 25, member 32 
                 SLC25A32 
                 NM_030780 
               
               
                 ferritin, heavy polypeptide pseudogene 1 
                 FTHP1 
                 GENSCAN00000008165 
               
               
                 ATPase, H+ transporting, lysosomal 50/57 kDa, V1 subunit H 
                 ATP6V1H 
                 NM_015941 
               
               
                 Histone cluster 1, H1e 
                 HIST1H1E 
                 NM_005321 
               
            
           
           
               
            
               
                 Table 3B 
               
            
           
           
               
               
               
            
               
                 4-aminobutyrate aminotransferase 
                 ABAT 
                 NM_020686 
               
               
                 abhydrolase domain containing 5 
                 ABHD5 
                 NM_016006 
               
               
                 alkaline ceramidase 2 
                 ACER2 
                 NM_001010887 
               
               
                 ATP citrate lyase 
                 ACLY 
                 NM_001096 
               
               
                 actin-related protein 10 homolog 
                 ACTR10 
                 NM_018477 
               
               
                 ADAM metallopeptidase domain 20 
                 ADAM20 
                 NM_003814 
               
               
                 Retrotransposed pseudogene AL391261.2-201 
                 AL391261.2-201 
                 GENSCAN00000063078 
               
               
                 aldehyde dehydrogenase 18 family, member A1 
                 ALDH18A1 
                 NM_002860 
               
               
                 aldehyde dehydrogenase 1 family, member B1 
                 ALDH1B1 
                 NM_000692 
               
               
                 alkB, alkylation repair homolog 6 ( E.   coli ) 
                 ALKBH6 
                 NM_032878 
               
               
                 anaphase promoting complex subunit 1 
                 ANAPC1 
                 NM_022662 
               
               
                 anaphase promoting complex subunit 5 
                 ANAPC5 
                 NM_016237 
               
               
                 ankyrin repeat, family A (RFXANK-like), 2 
                 ANKRA2 
                 NM_023039 
               
               
                 ADP-ribosylation factor GTPase activating protein 3 
                 ARFGAP3 
                 NM_014570 
               
               
                 Rho GTPase activating protein 9 
                 ARHGAP9 
                 NM_032496 
               
               
                 ankyrin repeat and SOCS box-containing 7 
                 ASB7 
                 NM_198243 
               
               
                 ATPase, H+ transporting, lysosomal 9 kDa, V0 subunit e1 
                 ATP6V0E1 
                 NM_003945 
               
               
                 bridging integrator 2 
                 BIN2 
                 NM_016293 
               
               
                 bleomycin hydrolase 
                 BLMH 
                 NM_000386 
               
               
                 brix domain containing 1/ribosome production factor 2 
                 BXDC1/RPF2 
                 ENST00000368864 
               
               
                 homolog 
                   
                   
               
               
                 chromosome 11 open reading frame 61 
                 C11orf61 
                 NM_024631 
               
               
                 chromosome 11 open reading frame 67 
                 C11orf67 
                 NM_024684 
               
               
                 chromosome 12 open reading frame 57 
                 C12orf57 
                 NM_138425 
               
               
                 chromosome 13 open reading frame 18 
                 C13orf18 
                 NM_025113 
               
               
                 chromosome 15 open reading frame 24 
                 C15orf24 
                 NM_020154 
               
               
                 chromosome 19 open reading frame 54 
                 C19orf54 
                 NM_198476 
               
               
                 chromosome 1 open reading frame 174 
                 C1orf174 
                 NM_207356 
               
               
                 chromosome 1 open reading frame 183 
                 C1orf183 
                 NM_019099 
               
               
                 chromosome 20 open reading frame 111 
                 C20orf111 
                 NM_016470 
               
               
                 chromosome 20 open reading frame 24 
                 C20orf24 
                 BC004446 
               
               
                 chromosome 3 open reading frame 62/ubiquitin specific  
                 C3orf62/USP4 
                 BC023586 
               
               
                 peptidase 4 (proto-oncogene) 
                   
                   
               
               
                 chromosome 9 open reading frame 89 
                 C9orf89 
                 BC038856 
               
               
                 coactivator-associated arginine methyltransferase 1 
                 CARM1 
                 NM_199141 
               
               
                 CD33 molecule 
                 CD33 
                 NM_001772 
               
               
                 CD86 molecule 
                 CD86 
                 NM_175862 
               
               
                 CD93 molecule 
                 CD93 
                 NM_012072 
               
               
                 cytochrome c oxidase subunit VIIa polypeptide 2 like 
                 COX7A2L 
                 NM_004718 
               
               
                 corticotropin releasing hormone binding protein 
                 CRHBP 
                 NM_001882 
               
               
                 chondroitin sulfate N-acetylgalactosaminyltransferase 2 
                 CSGALNACT2 
                 NM_018590 
               
               
                 Cytochrome P450 51A1 
                 CYP51A1 
                 NM_000786.2 
               
               
                 DDRGK domain containing 1 
                 DDRGK1 
                 NM_023935 
               
               
                 DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 
                 DDX21 
                 NM_004728 
               
               
                 24-dehydrocholesterol reductase 
                 DHCR24 
                 NM_014762 
               
               
                 7-dehydrocholesterol reductase 
                 DHCR7 
                 NM_001360 
               
               
                 DEAH (Asp-Glu-Ala-His) box polypeptide 33 
                 DHX33 
                 NM_020162 
               
               
                 DnaJ (Hsp40) homolog, subfamily B, member 4 
                 DNAJB4 
                 NM_007034 
               
               
                 DnaJ (Hsp40) homolog, subfamily B, member 9 
                 DNAJB9 
                 NM_012328 
               
               
                 DnaJ (Hsp40) homolog, subfamily C, member 5 
                 DNAJC5 
                 NM_025219 
               
               
                 DnaJ (Hsp40) homolog, subfamily C, member 9 
                 DNAJC9 
                 NM_015190 
               
               
                 D-tyrosyl-tRNA deacylase 1 homolog 
                 DTD1 
                 NM_080820 
               
               
                 ER degradation enhancer, mannosidase alpha-like 2 
                 EDEM2 
                 NM_018217 
               
               
                 ecotropic viral integration site 2B 
                 EVI2B 
                 NM_006495 
               
               
                 family with sequence similarity 36, member A 
                 FAM36A 
                 NM_198076 
               
               
                 family with sequence similarity 86, member A 
                 FAM86A 
                 NM_201400 
               
               
                 Fas (TNF receptor superfamily, member 6) 
                 FAS 
                 NM_000043 
               
               
                 MGC44478 
                 FDPSL2A 
                 NR_003262 
               
               
                 ferredoxin reductase 
                 FDXR 
                 NM_024417 
               
               
                 forkhead box O4 
                 FOXO4 
                 NM_005938 
               
               
                 FTHL10-001, Transcribed processed pseudogene 
                 FTHL10-001 
                 NR_002200 
               
               
                 fucosidase, alpha-L-2, plasma 
                 FUCA2 
                 NM_032020 
               
               
                 growth arrest-specific 2 like 3 
                 GAS2L3 
                 NM_174942 
               
               
                 ganglioside induced differentiation associated protein 2  
                 GDAP2 
                 NM_017686 
               
               
                 growth differentiation factor 11 
                 GDF11 
                 NM_005811 
               
               
                 glutaredoxin (thioltransferase) 
                 GLRX 
                 NM_002064 
               
               
                 guanine nucleotide binding protein-like 3 
                 GNL3L 
                 NM_019067 
               
               
                 glucosamine-phosphate N-acetyltransferase 1 
                 GNPNAT1 
                 NM_198066 
               
               
                 glutathione reductase 
                 GSR 
                 NM_000637 
               
               
                 GTF2I repeat domain containing 213/2/2 pseudogene 
                 GTF2IRD2B/2/2P 
                 BC067859 
               
               
                 general transcription factor IIIC, polypeptide 2 beta 
                 GTF3C2 
                 NM_001521 
               
               
                 HMG-box transcription factor 1 
                 HBP1 
                 NM_012257 
               
               
                 histone cluster 1, H1c 
                 HIST1H1C 
                 NM_005319 
               
               
                 histone cluster 1, H2ae 
                 HIST1H2AE 
                 NM_021052 
               
               
                 histone cluster 1, H2be 
                 HIST1H2BE 
                 NM_003523 
               
               
                 histone clusters 1, H2bm/2, H3, pseudogene 2/2, H2b/a 
                 HIST1H2BM/ 
                 NM_001024599 
               
               
                   
                 HIST2H3PS2/BF/A 
                   
               
               
                 histone cluster 1, H3g 
                 HIST1H3G 
                 NM_003534 
               
               
                 histone cluster 1, H3j 
                 HIST1H3J 
                 NM_003535 
               
               
                 histone cluster 1, H4a 
                 HIST1H4A 
                 NM_003538 
               
               
                 histone clusters 2, H2aa3/2, H2aa4 
                 HIST2H2AA3/4 
                 NM_003516 
               
               
                 high-mobility group box 3 
                 HMGB3 
                 NM_005342 
               
               
                 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 
                 HMGCR 
                 NM_000859 
               
               
                 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 
                 HMGCS1 
                 NM_001098272 
               
               
                 heme oxygenase (decycling) 1 
                 HMOX1 
                 NM_002133 
               
               
                 heterogeneous nuclear ribonucleoprotein L 
                 HNRNPL 
                 NM_001533 
               
               
                 insulin receptor substrate 2 
                 IRS2 
                 NM_003749 
               
               
                 iron-sulfur cluster scaffold homolog 
                 ISCU 
                 NM_014301 
               
               
                 interferon stimulated exonuclease gene 20 kDa-like 2 
                 ISG20L2 
                 NM_030980 
               
               
                 potassium voltage-gated channel, Isk-related family, member 3 
                 KCNE3 
                 NM_005472 
               
               
                 hypothetical protein LOCI 00132855/ATPase, H+ transporting, 
                 LOC100132855/ 
                 NM_004691 
               
               
                 lysosomal 38 kDa, V0 subunit d1 
                 ATP6V0D1 
                   
               
               
                 hCG1651476 
                 LOC284417 
                 NM_001085488 
               
               
                 golgi autoantigen, golgin subfamily a, 6 pseudogene/ 
                 LOC729668/ 
                 NM_018109 
               
               
                 mitochondrial poly(A) polymerase 
                 MTPAP 
                   
               
               
                 lysophosphatidic acid receptor 1 
                 LPAR1 
                 NM_057159 
               
               
                 leucine-rich PPR-motif containing 
                 LRPPRC 
                 NM_133259 
               
               
                 lymphocyte antigen 96 
                 LY96 
                 NM_015364 
               
               
                 mitogen-activated protein kinase kinase 1 
                 MAP2K1 
                 NM_002755 
               
               
                 mitogen-activated protein kinase 13 
                 MAPK13 
                 NM_002754 
               
               
                 methyltransferase like 2A 
                 METTL2A 
                 NM_181725 
               
               
                 Brain cDNA clone: similar to human METTL2 
                 METTL2B 
                 NM_018396.1 
               
               
                 Methyltransferase like 2B 
                 METTL2B 
                 NM_018396.2 
               
               
                 microsomal glutathione S-transferase 3 
                 MGST3 
                 NM_004528 
               
               
                 mitochondrial ribosomal protein L30 
                 MRPL30 
                 NM_145212 
               
               
                 mitochondrial ribosomal protein L4 
                 MRPL4 
                 NM_146388 
               
               
                 mitochondrial ribosomal protein S17 
                 MRPS17 
                 NM_015969 
               
               
                 5-methyltetrahydrofolate-homocysteine methyltransferase 
                 MTR 
                 NM_000254 
               
               
                 MYB binding protein (P160) 1a 
                 MYBBP1A 
                 NM_014520 
               
               
                 neighbor of BRCA1 gene 1 
                 NBR1 
                 NM_031858 
               
               
                 nuclear import 7 homolog 
                 NIP7 
                 NM_016101 
               
               
                 NLR family, pyrin domain containing 12 
                 NLRP12 
                 NM_144687 
               
               
                 nucleolar protein family 6 (RNA-associated) 
                 NOL6 
                 NM_022917 
               
               
                 NAD(P)H dehydrogenase, quinone 1 
                 NQO1 
                 NM_000903 
               
               
                 nuclear receptor binding protein 1 
                 NRBP1 
                 NM_013392 
               
               
                 nucleotide binding protein-like 
                 NUBPL 
                 NM_025152 
               
               
                 nudix (nucleoside diphosphate linked moiety X)-type motif 14 
                 NUDT14 
                 NM_177533 
               
               
                 nuclear fragile X mental retardation protein interacting  
                   
                   
               
               
                 protein 1 
                 NUFIP1 
                 NM_012345 
               
               
                 nucleoporin 153 kDa 
                 NUP153 
                 NM_005124 
               
               
                 olfactory receptor, family 5, subfamily B, member 21 
                 OR5B21 
                 NM_001005218 
               
               
                 PAS domain containing serine/threonine kinase 
                 PASK 
                 NM_015148 
               
               
                 PRKC, apoptosis, WT1, regulator 
                 PAVVR 
                 NM_002583 
               
               
                 PDGFA associated protein 1 
                 PDAP1 
                 NM_014891 
               
               
                 phosphodiesterase 1B, calmodulin-dependent 
                 PDE1B 
                 NM_000924 
               
               
                 phosphoribosylformylglycinamidine synthase 
                 PFAS 
                 NM_012393 
               
               
                 pleckstrin homology-like domain, family A, member 3 
                 PHLDA3 
                 NM_012396 
               
               
                 phosphoinositide-3-kinase adaptor protein 1 
                 PIK3AP1 
                 NM_152309 
               
               
                 PTEN induced putative kinase 1 
                 PINK1 
                 NM_032409 
               
               
                 phosphomannomutase 2 
                 PMM2 
                 NM_000303 
               
               
                 partner of NOB1 homolog 
                 PNO1 
                 NM_020143 
               
               
                 polymerase (RNA) II (DNA directed) polypeptide E, 25 kDa 
                 POLR2E 
                 NM_002695 
               
               
                 polymerase (RNA) III (DNA directed) polypeptide E (80 kD)  
                 POLR3E 
                 NM_018119 
               
               
                 protein phosphatase 1D magnesium-dependent, delta isoform 
                 PPM1D 
                 BC042418 
               
               
                 phosphatidylinositol-3,4,5-trisphosphate-dependent Rac 
                 PREX1 
                 NM_020820 
               
               
                 exchange factor 1 
                   
                   
               
               
                 proline-serine-threonine phosphatase interacting protein 1 
                 PSTPIP1 
                 NM_003978 
               
               
                 prothymosin, alpha 
                 PTMA 
                 NM_016184/024809 
               
               
                 RAB33B, member RAS oncogene family 
                 RAB33B 
                 NM_031296 
               
               
                 renin binding protein 
                 RENBP 
                 NM_002910 
               
               
                 replication factor C (activator 1) 2, 40 kDa 
                 RFC2 
                 NM_181471 
               
               
                 ribonuclease H1 
                 RNASEH1 
                 NM_002936 
               
               
                 ring finger protein 146 
                 RNF146 
                 NM_030963 
               
               
                 ring finger protein 24 
                 RNF24 
                 NM_007219 
               
               
                 ring finger protein 26 
                 RNF26 
                 NM_032015 
               
               
                 Havana pseudogene RP1-274L14.2-001 
                 RP1-274L14.2-001 
                 NM_032020 
               
               
                 ribosomal protein SA/small nucleolar RNA, H/ACA box 62 
                 RPSA/SNORA62 
                 NM_014570 
               
               
                 RNA pseudouridylate synthase domain containing 2 
                 RPUSD2 
                 NM_152260 
               
               
                 ribosomal RNA processing 12 homolog 
                 RRP12 
                 NM_015179 
               
               
                 retinoid X receptor, alpha 
                 RXRA 
                 NM_002957 
               
               
                 scavenger receptor class B, member 2 
                 SCARB2 
                 NM_005506 
               
               
                 SERPINE1 mRNA binding protein 1 
                 SERBP1 
                 NM_001018067 
               
               
                 splicing factor proline/glutamine-rich 
                 SFPQ 
                 NM_005066 
               
               
                 solute carrier family 35, member B3 
                 SLC35B3 
                 BX538271 
               
               
                 solute carrier family 37, member 4 
                 SLC37A4 
                 NM_001467 
               
               
                 solute carrier family 5, member 6 
                 SLC5A6 
                 NM_021095 
               
               
                 sphingomyelin phosphodiesterase 4, neutral membrane 
                 SMPD4 
                 NM_017751 
               
               
                 small nucleolar(sn)RNA host gene 1, non-coding/snRNA C/D  
                 SNHG1/SNORD26 
                 NM_002032 
               
               
                 box 26 
                   
                   
               
               
                 small nucleolar RNA host gene 12 (non-coding) 
                 SNHG12 
                 NM_207356 
               
               
                 small nucleolar RNA, H/ACA box 45 
                 SNORA45 
                 NR_002977 
               
               
                 SnRNA 
                 SnRNA 
                 Affymetrix: 7966223 
               
               
                 sorting nexin family member 27 
                 SNX27 
                 NM_030918 
               
               
                 sterol regulatory element binding transcription factor 2  
                 SREBF2 
                 NM_004599 
               
               
                 RRNA SSU_rRNA_5 
                 SSU_rRNA_5 
                 ENST00000386723 
               
               
                 ST3 beta-galactoside alpha-2,3-sialyltransferase 6 
                 ST3GAL6 
                 NM_006100 
               
               
                 serine/threonine kinase 17b 
                 STK17B 
                 NM_004226 
               
               
                 tubulin folding cofactor E-like 
                 TBCEL 
                 NM_152715 
               
               
                 tectonic family member 2 
                 TCTN2 
                 NM_024809 
               
               
                 toll-like receptor 6 
                 TLR6 
                 NM_006068 
               
               
                 toll-like receptor 9/twinfilin homolog 2 
                 TLR9/TWF2 
                 NM_007284 
               
               
                 transmembrane protein 55A 
                 TMEM55A 
                 NM_018710 
               
               
                 transmembrane protein 59 
                 TMEM59 
                 NM_004872 
               
               
                 transmembrane protein 77 
                 TMEM77 
                 BC091509 
               
               
                 transmembrane protein 97 
                 TMEM97 
                 NM_014573 
               
               
                 tumor necrosis factor receptor superfamily, member 10c 
                 TNFRSF10C 
                 NM_003841 
               
               
                 translocase of outer mitochondrial membrane 34 
                 TOMM34 
                 NM_006809 
               
               
                 translocase of outer mitochondrial membrane 40 homolog 
                 TOMM40 
                 BC001779 
               
               
                 translocase of outer mitochondrial membrane 5 homolog /F- 
                   
                   
               
               
                 box protein 10 
                 TOMM5/FBXO10 
                 NM_012166 
               
               
                 tumor protein p53 inducible protein 3 
                 TP53I3 
                 NM_004881 
               
               
                 tumor protein p53 inducible nuclear protein 1 
                 TP53INP1 
                 NM_033285 
               
               
                 thioredoxin reductase 1 
                 TXNRD1 
                 NM_003330 
               
               
                 ubiquitin-fold modifier conjugating enzyme 1 
                 UFC1 
                 NM_016406 
               
               
                 ubiquitin specific peptidase 10 
                 USP10 
                 NM_005153 
               
               
                 vesicle-associated membrane protein 3 (cellubrevin) 
                 VAMP3 
                 NM_004781 
               
               
                 valyl-tRNA synthetase 
                 VARS 
                 NM_006295 
               
               
                 vacuolar protein sorting 37 homolog A 
                 VPS37A 
                 NM_152415 
               
               
                 zinc finger protein 211 
                 ZNF211 
                 NM_006385 
               
               
                 zinc finger protein 223 
                 ZNF223 
                 NM_013361 
               
               
                 zinc finger protein 561 
                 ZNF561 
                 NM_152289 
               
               
                 zinc finger protein 79 
                 ZNF79 
                 NM_007135 
               
               
                   
               
            
           
         
       
     
     Table 3 legend. The table shows the profile genes found by t-test and Backward Elimination. Genes were annotated, using the NetAffx database from Affymetrix (www.affymetrix.com, Santa Clara USA). When found, the Unigene (www.ncbi.nlm.nih.gov/UniGene/) ID was chosen as the gene identifier. In the twelve cases where no Unigene ID was reported the best alternative ID was given. Gene names and IDs were checked against the IPA database where 189 of the 200 could be matched. In one instance only an Affymetrix ID was reported. 6 duplicate genes were removed. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Dominating functions in the “Prediction signature”. 184 of the 200 molecules 
               
               
                 were investigated functionally using IPA. Only functions populated by 15 or more 
               
               
                 molecules were reported. 
               
            
           
           
               
               
               
               
            
               
                   
                 Number  
                   
                   
               
               
                   
                 of 
                   
                   
               
               
                   
                 molecules 
                   
                   
               
               
                   
                 from 
                   
                 Most prominent 
               
               
                 Function 
                 signature 
                 Molecule names 
                 sub functions 
               
               
                   
               
               
                 small  
                 38 
                 ABHD5, ACLY, ALDH18A1, BLMH, CD86, 
                 Metabolism (23), 
               
               
                 molecule 
                   
                 CSGALNACT2, CYP51A1, DHCR24, DHCR7, DNAJC5, 
                 biosynthesis (14), 
               
               
                 biochemistry 
                   
                 FAS, FASN, FDXR, GLRX, GNPNAT1, HMGCR, 
                 modification (12), 
               
               
                   
                   
                 HMOX1, IRS2, LPAR1, LY96, MGST3, MTR, NQO1, 
                 synthesis (10) 
               
               
                   
                   
                 PASK, PDE1B, PINK1, PMM2, RENBP, RXRA, 
                   
               
               
                   
                   
                 SLC25A32, SLC37A4, SLC5A6, SMPD4, SQLE, 
                   
               
               
                   
                   
                 SREBF2, ST3GAL6, TLR6, TMEM55A 
                   
               
               
                 cell death 
                 33 
                 CD33, DDX19A, DHCR24, DNAJB9, DNAJC5, FAS, 
                 Apoptosis (30),  
               
               
                   
                   
                 FASN, FDXR, FOXO4, GLRX, GNPNAT1, GSR, 
                 cell death (13) 
               
               
                   
                   
                 HIST1H1C, HMGB3, HMOX1, IRS2, LPAR1, MAP2K1, 
                   
               
               
                   
                   
                 MAPK13, NQO1, PAWR, PDE1B, PHLDA3, PINK1, 
                   
               
               
                   
                   
                 PPM1D, RXRA, SERBP1, SPRY2, STK17B, TLR6, 
                   
               
               
                   
                   
                 TNFRSF10C, TP53INP1, TXNRD1 
                   
               
               
                 lipid 
                 24 
                 ABHD5, ACLY, CYP51A1, DHCR24, DHCR7, FAS, 
                 Metabolism (17), 
               
               
                 metabolism 
                   
                 FASN, FDXR, HMGCR, HMOX1, IRS2, LPAR1, LY96, 
                 modification (11), 
               
               
                   
                   
                 MGST3, PASK, RENBP, RXRA, SLC37A4, SMPD4, 
                 synthesis (10) 
               
               
                   
                   
                 SQLE, SREBF2, ST3GAL6, TLR6, TMEM55A 
                   
               
               
                 hemato- 
                 19 
                 CARM1, CD33, CD86, FAS, FOXO4, HIST1H1C, 
                 Proliferation (10), 
               
               
                 logical 
                   
                 HMGB3, HMGCR, HMOX1, IRS2, LY96, NBR1, NQO1, 
                 apoptosis (5) 
               
               
                 system 
                   
                 PAWR, PIK3AP1, PPM1D, STK17B, TP53INP1, VAMP3 
                   
               
               
                 development 
                   
                   
                   
               
               
                 cellular  
                 16 
                 CARM1, CD33, CD86, FAS, FOXO4, HIST1H1C, 
                 Proliferation (16), 
               
               
                 growth and 
                   
                 HMGB3, HMGCR, HMOX1, IRS2, LY96, NBR1, NQO1, 
                 growth (4) 
               
               
                 proliferation 
                   
                 PAWR, PIK3AP1, PPM1D, STK17B, TP53INP1, VAMP3 
                   
               
               
                 molecular 
                 15 
                 CARM1, CD33, CD86, FAS, FOXO4, HIST1H1C, 
                 Accumulation (8), 
               
               
                 transport 
                   
                 HMGB3, HMGCR, HMOX1, IRS2, LY96, NBR1, NQO1, 
                 quantity (5) 
               
               
                   
                   
                 PAWR, PIK3AP1, PPM1D, STK17B, TP53INP1, VAMP3 
                   
               
               
                 cell cycle 
                 15 
                 DNAJB4, DTD1, FAS, FASN, FOXO4, GDF11, HBP1, 
                 Cell cycle 
               
               
                   
                   
                 HMOX1, IRS2, MAP2K1, PAWR, PPM1D, SFPQ, 
                 progression (13), 
               
               
                   
                   
                 SPRY2, TP53INP1 
                 cell division (5) 
               
               
                 carbohydrate 
                 15 
                 DNAJB4, DTD1, FAS, FASN, FOXO4, GDF11, HBP1, 
                 Metabolism (9), 
               
               
                 metabolism 
                   
                 HMOX1, IRS2, MAP2K1, PAWR, PPM1D, SFPQ, 
                 biosynthesis (5) 
               
               
                   
                   
                 SPRY2, TP53INP1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Support Vector Machine (SVM) algorithm 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 filnamnTraining &lt;-“training set.txt” 
               
               
                 filnamnTest &lt;-“test set.txt” 
               
               
                 lista &lt;- read.delim(“biomarker signature.txt”, 
               
               
                 header=FALSE) # EN FIL 
               
               
                 MED DE ANALYTER 
               
               
                 lista &lt;- as.character(lista[[1]]) 
               
               
                 listaBoolean &lt;- is.element(ProteinNames, lista) 
               
               
                 group1&lt;- “pos” 
               
               
                 group2&lt;- “neg” 
               
               
                 rawfile &lt;- read.delim(filnamnTraining) 
               
               
                 samplenames &lt;- as.character(rawfile[,1] ) 
               
               
                 groupsTraining &lt;- rawfile[,2] 
               
               
                 dataTraining &lt;- t(rawfile[,-c(1,2) ] ) 
               
               
                 ProteinNames &lt;- read.delim(filnamn 
               
               
                 Training,header=FALSE) 
               
               
                 ProteinNames &lt;- as.character(as.matrix 
               
               
                 (ProteinNames)[1,]) 
               
               
                 ProteinNames &lt;- ProteinNames[-(1:2) ] 
               
               
                 listaBoolean &lt;- is.element(ProteinNames, lista) 
               
               
                 rownames(dataTraining) &lt;- ProteinNames 
               
               
                 colnames(dataTraining) &lt;- samplenames 
               
               
                 logdataTraining &lt;- dataTraining 
               
               
                 logdataTraining &lt;- logdataTraining[listaBoolean, ] 
               
               
                 rawfile &lt;- read.delim(filnamnTest) 
               
               
                 samplenames &lt;- as.character(rawfile[,1] ) 
               
               
                 groupsTest &lt;- rawfile[,2] 
               
               
                 dataTest &lt;- t(rawfile[,-c(1,2) ] ) 
               
               
                 ProteinNames &lt;- read.delim(filnamn 
               
               
                 Test,header=FALSE) 
               
               
                 ProteinNames &lt;- as.character(as.matrix 
               
               
                 (ProteinNames)[1 ,] ) 
               
               
                 ProteinNames &lt;- ProteinNames[-(1:2) ] 
               
               
                 rownames(dataTest) &lt;- ProteinNames 
               
               
                 colnames(dataTest) &lt;- samplenames 
               
               
                 logdataTest &lt;-dataTest 
               
               
                 logdataTest &lt;- logdataTest[listaBoolean, ] 
               
               
                 svmfacTraining&lt;- factor(rep(‘rest’,ncol(logdataTraining)), 
               
               
                 levels=c(group1, group2, ‘rest’)) 
               
               
                 subset1Training&lt;- is.element(groupsTraining , 
               
               
                 strsplit(group1,“,”)[ [ 1 ] ] ) 
               
               
                 subset2Training&lt;- is.element(groupsTraining , 
               
               
                 strsplit(group2,“,”)[ [ 1 ] ] ) 
               
               
                 svmfacTraining[subset1Training] &lt;- group1 
               
               
                 svmfacTraining[subset2Training] &lt;- group2 
               
               
                 facTraining &lt;-factor(as.character(svmfacTraining 
               
               
                 [subset1Training|subset2Training] ), 
               
               
                 levels=c(group1,group2) ) 
               
               
                 svmfacTest&lt;- factor(rep(‘rest’,ncol (logdataTest ) ), 
               
               
                 levels=c(group1, group2, ‘rest’) ) 
               
               
                 subset1Test&lt;- is.element(groupsTest , strsplit 
               
               
                 (group1,“,”) [ [ 1 ] ] ) 
               
               
                 subset2Test&lt;- is.element(groupsTest , strsplit 
               
               
                 (group2,“,”) [ [ 1 ] ] ) 
               
               
                 svmfacTest[subset1Test] &lt;- group1 
               
               
                 svmfacTest[subset2Test] &lt;- group2 
               
               
                 facTest &lt;-factor (as.character(svmfacTest 
               
               
                 [subset1TestIsubset2Test ] ),levels=c(group1,group2) ) 
               
               
                 n1 &lt;- sum(facTest ==levels(facTest ) [1] ) 
               
               
                 n2 &lt;- sum(facTest ==levels(facTest ) [2] ) 
               
               
                 nsamples &lt;- n1+n2 
               
               
                 SampleInformation &lt;- paste(levels(facTest ) 
               
               
                 [ 1 ],“ ”,n1, “ , ”, levels(facTest) [2],“ ”,n2,sep=“”) 
               
               
                 svmtrain &lt;- svm(t (logdataTraining) , fac 
               
               
                 Training , kernel=“linear” ) 
               
               
                 pred&lt;-predict (svmtrain , t(logdataTest) , 
               
               
                 decision.values=TRUE) 
               
               
                 res&lt;-attr(pred, “decision.values”) 
               
               
                 names &lt;- colnames(logdataTest, do.NULL=FALSE) 
               
               
                 orden &lt;- order(res , decreasing=TRUE) 
               
               
                 Samples &lt;- data.frame(names [orden] , 
               
               
                 res [orden] , facTest [orden] ) 
               
               
                 ROCdata &lt;- myROC(res,facTest) 
               
               
                 SenSpe &lt;- SensitivitySpecificity(res,facTest) 
               
               
                 ROCplot(list(SampleInformation=SampleInformation, 
               
               
                 ROCarea=ROCdata [1] ,p.value=ROCdata [2], 
               
               
                 SenSpe &lt;- SenSpe,samples=Samples), 
               
               
                 sensspecnumber=4) 
               
               
                 #rows in blue are needed only for ROC evaluation. 
               
               
                 #to assess an unknown sample, print res. 
               
               
                 #(vector with prediction values)