Methods to Diagnose Type 1 Diabetes by measuring cytokine and/or chemokine expression profiles.

Field of the Invention

[0001] The invention relates to methods for detecting and diagnosing type 1 diabetes or detecting an increased likelihood of developing type 1 diabetes using cytokine and chemokine expression profiles. Background of the Invention

[0002] Cytokines are proteins released by activated cells. They have a critical involvement in all inflammatory diseases/conditions, and as such are the ultimate effectors of a biological process. Cytokines are categorized as pro-inflammatory (important in causing and maintaining inflammation), anti- inflammatory (important to reduce the level of inflammation), and chemokines (cytokines that function as chemoattractants to recruit cells to sites of inflammation).

[0003] Cytokines have a critical involvement in the development and progression of type 1 diabetes. Anatomically located in the pancreas is the 'islets of Langerhans' that contain insulin-secreting beta cells. Type 1 diabetes is an autoimmune disease that arises from the specific destruction of beta cells by T cells resulting in impaired insulin secretion and corresponding hypoglycemia.

[0004] In normal inflammatory situations, dendritic cells are critical for activating T cells particularly during times of infection whereby T cells are required to clear disease-causing pathogens. In type 1 diabetes, dendritic cells are thought to orchestrate the autoimmune inflammatory process by abnormally activating T cells to attack self beta cells. Inflammation involves the recruitment of several cell types to the islets of Langerhans (via chemokines) followed by their activation (via cytokines). Each infiltrating cell releases a number of different cytokines and chemokines, thus generating a cytokine "reservoir".

Summary of the Invention

[0005] The inventors have analyzed the expression of multiple cytokines and chemokines in subjects with type 1 diabetes and have found a distinct pattern of cytokine and chemokine expression in subjects with type 1 diabetes as compared to normal, non-diabetic controls. Specifically, the inventors found significantly lower levels of the cytokines IL-8 and IL-1 alpha and of the chemokine MIP-1 beta in subjects with type 1 diabetes as compared to normal, non-diabetic controls. The inventors also found lower levels of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha, MIP-1 beta, and MIG, and increased levels of RANTES in subjects with type 1 diabetes as compared to normal, non-diabetic controls. This distinct profile of cytokine and chemokine expression can be used to detect or diagnose type 1 diabetes or identify subjects with an increased likelihood of developing type 1 diabetes.

[0006] Accordingly, in one embodiment the invention includes methods of detecting or diagnosing type 1 diabetes in a subject or detecting an increased likelihood of developing type 1 diabetes in a subject comprising: (a) determining the expression of one or more cytokine or chemokine from a test sample from a subject to generate a subject expression profile, wherein the cytokine comprises IL-8, IL-1 alpha or TNF-alpha and wherein the chemokine comprises MIP-1 alpha, MIP-1 beta, RANTES or MIG; and (b) comparing the subject expression profile to a control, wherein a difference in the subject expression profile as compared to the control is indicative of type 1 diabetes or an increased likelihood of developing type 1 diabetes in the subject.

[0007] Another embodiment includes the use of a cytokine or chemokine expression profile for detecting or diagnosing type 1 diabetes in a subject, or detecting an increased likelihood of developing type 1 diabetes in a subject wherein the cytokine comprises IL-8, IL-1 alpha or TNF-alpha and the chemokine comprises MIP-1 alpha, MIP-1 beta, RANTES or MIG. In a further embodiment, a difference in the expression profile of a subject compared to a control expression profile is indicative of type 1 diabetes or an increased likelihood of developing type 1 diabetes in the subject.

[0008] In some embodiments, the subject is human. In some embodiments the cytokine comprises IL-8, and the IL-8 expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In some embodiments the cytokine comprises IL-1 alpha, and the level of IL-1 alpha expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In some embodiments the cytokine comprises TNF-alpha and the level of TNF-alpha expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In another embodiment the chemokine comprises MIP-1 alpha, and the level of MIP-1 alpha expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In other embodiments the chemokine comprises MIP-1 beta and the level of MIP-1 beta expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes.

[0009] In some embodiments the cytokine comprises IL-8, IL-1 alpha and TNF-alpha and the chemokine comprises MIP-1 alpha and MIP-1 beta and the IL-8 levels are lower, the IL-1 levels are lower, the TNF-alpha levels are lower, the MIP-1 alpha levels are lower and the MIP-1 beta levels are lower as compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing diabetes. In a further embodiment, the IL-8 levels are lower, IL-1 alpha levels are lower, TNF-alpha levels are lower, MIP-1 alpha levels are lower, MIP-1 beta levels are lower, MIG levels are lower and/or RANTES levels are higher as compared to the control indicative of type 1 diabetes in the subject or an increased liklihood of developing diabetes. [0010] In some embodiments, the expression of the cytokine or chemokine is determined using protein based protocols. In another

embodiment, the expression of the cytokine or chemokine is determined using an antibody or antibody fragment directed against the cytokine or chemokine. Additionally, the antibody or antibody fragment may be labeled with a detectable marker. In further embodiments, the expression of the cytokine or chemokine is determined using multiplexing technology.

[0011] In some embodiments the test sample is selected from the group consisting of whole blood, plasma and serum. In one embodiment the test sample is plasma or serum.

[0012] In one embodiment, the expression of the cytokine or chemokine is determined by measuring or detecting RNA transcripts. In some embodiments, the step of determining the expression comprises RT-PCR or quantitative RT-PCR of the test sample. In another embodiment, the step of determining expression comprises microarray analysis of the test sample. In some embodiments, the RT-PCR is conducing using multiplexing technology. [0013] The inventors also analyzed the levels of multiple cytokines and chemokines secreted by blood dendritic cells from subjects with type 1 diabetes and have found a distinct pattern of cytokine and chemokine expression in subjects with type 1 diabetes as compared to normal, non- diabetic controls. Specifically, the inventors found a significantly higher level of the chemokine RANTES in subjects with type 1 diabetes as compared to normal, non-diabetic controls. The inventors also found lower production of IL- 6, IL-8, IL-1 alpha, IL-12p70, TNF-alpha, MIP-1 alpha, and MIP-1 beta and increased production of RANTES and MCP-1 by blood dendritic cells in subjects with type 1 diabetes as compared to normal, non-diabetic controls. This distinct cytokine and chemokine expression profile that is secreted by blood dendritic cells can be used to detect or diagnose type 1 diabetes or detecting an increased likelihood of developing type 1 diabetes in a subject.

[0014] Accordingly, in one embodiment the invention includes a method of detecting or diagnosing type 1 diabetes in a subject or detecting an increased likelihood of developing type 1 diabetes in a subject comprising: (a) determining the expression of one or more cytokine or chemokine secreted by

blood dendritic cells from a subject to generate a subject expression profile, wherein the cytokine comprises IL-6, IL-8, IL-1 alpha, IL-12p70, or TNF-alpha and wherein the chemokine comprises MIP-1 alpha, MIP-1 beta, RANTES or MCP-1 ; and (b) comparing the subject expression profile to a control, wherein a difference in the subject expression profile as compared to the control is indicative of type 1 diabetes or an increased likelihood of developing type 1 diabetes in the subject.

[0015] Another embodiment includes the use of a cytokine or chemokine profile secreted by blood dendritic cells for detecting or diagnosing type 1 diabetes in a subject or detecting an increased likelihood of developing type 1 diabetes in a subject wherein the cytokine comprises IL-6, IL-8, IL-1 alpha, IL-12p70, or TNF-alpha and the chemokine comprises MIP-1 alpha, MIP-1 beta, RANTES or MCP-1. In a further embodiment, a difference in the in the expression profile of cytokines or chemokines secreted by the blood dendritic cells of a subject as compared to a control expression profile is indicative of type 1 diabetes or an increased likelihood of developing type 1 diabetes in the subject.

[0016] In some embodiments, the subject is human. In additional embodiments, the chemokine comprises RANTES and the level of RANTES expression is higher in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing diabetes. In another embodiment the cytokine comprises IL-6 and the level of IL-6 expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing diabetes. In another embodiment the cytokine comprises IL-8 and the level of IL-8 expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing diabetes. In still a further embodiment, the chemokine comprises MIP-1 alpha and the level of MIP-1 alpha expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing diabetes. In another embodiment the chemokine comprises MIP-1

beta and the level of MIP-1 beta expression is lower in the subject compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing diabetes.

[0017] In a further embodiment, the IL-6 levels are lower, IL-8 levels are lower, MIP-1 alpha levels are lower, MIP-1 beta levels are lower, RANTES levels are higher levels as compared to the control indicative of type 1 diabetes in the subject or an increased likelihood of type 1 diabetes in the subject.

[0018] In further embodiments, the IL-6 levels are lower, IL-8 levels are lower, IL-1 alpha levels are lower, IL-12p70 levels are lower, TNF-alpha levels are lower, MIP-1 alpha levels are lower, MIP-1 beta levels are lower, RANTES levels are higher and/or MCP-1 levels are higher as compared to the control indicative of type 1 diabetes in the subject or an increased likelihood of type 1 diabetes in the subject. [0019] In some embodiments the expression of the cytokine or chemokine is determined using protein based protocols. The expression of the cytokine or chemokine may be also determined using an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment is labeled with a detectable marker. The expression of the cytokine or chemokine may also be determined using multiplexing technology.

[0020] In some embodiments, the dendritic cells are in cell culture. In additional embodiments, the step of determining the expression comprises the analysis of cell culture media.

[0021] The invention also includes kits for detecting or diagnosing type 1 diabetes comprising detection agents for cytokines or chemokines according to the methods of the invention, and instructions for the use thereof. In some embodiments of the kit, the expression of the cytokine or chemokine is determined using protein based protocols. In other embodiments of the kit, the expression of the cytokine or chemokine is determined using nucleic acid

based protocols. In a further aspect, the invention includes kits for detecting the likelihood of developing type 1 diabetes in a subject.

[0022] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Brief Description of the Drawings

[0023] Embodiments of the invention will now be described in relation to the drawings in which:

[0024] Figure 1 shows the plasma levels of the pro-inflammatory cytokines IL-8 (A) and IL-1 alpha (B) in type 1 diabetes subjects (triangles) as compared to non-diabetic controls (squares).

[0025] Figure 2 shows the plasma levels of the pro-inflammatory cytokine TNF-alpha (E) and the chemokines MIP-1 beta (A), RANTES (B), MIG (C), and MIP-1 alpha (D) in type 1 diabetes subjects (triangles) as compared to non-diabetic controls (squares). [0026] Figure 3 shows the level of the chemokine RANTES secreted by dendritic cells from type 1 diabetes subjects (triangles) as compared to non- diabetic controls (squares).

[0027] Figure 4 shows the level of the pro-inflammatory cytokines IL-6

(A), IL-8 (B), IL-1 alpha (C), IL-12p70 (D) and TNF-alpha (E) secreted by dendritic cells from type 1 diabetes subjects (triangles) as compared to non- diabetic controls (squares).

[0028] Figure 5 shows the level of the chemokines MIP-1 alpha (A),

MIP-1 beta (B) and MCP-1 (C) secreted by dendritic cells from type 1 diabetes subjects (triangles) as compared to non-diabetic controls (squares).

[0029] Figure 6 shows a significant difference (p = 0.026) in the serum levels of the pro-inflammatory cytokine IL-8 in a separate group of subjects with type 1 diabetes (triangles) (n=13) compared to non-diabetic controls (squares) (n=13) (Figure 6A), wherein the subjects are age-matched and sex- matched (Figure 6B)

Detailed Description of the Invention

[0030] The inventors have found a distinct pattern of cytokine and chemokine expression in subjects with type 1 diabetes as compared to normal, non-diabetic controls. Specifically, the inventors found significantly lower levels of the cytokines IL-8 and IL-1 alpha and of the chemokine MIP-1 beta in subjects with type 1 diabetes as compared to normal, non-diabetic controls. In one aspect of the invention, the inventors found lower levels of IL- 1-alpha, IL-8, TNF-alpha, MIP-1-alpha and MIP-1-beta in the serum or plasma of subjects with type 1 diabetes compared to normal, non-diabetic controls. The inventors also found lower levels of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha, MIP-1 beta, and MIG, and increased levels of RANTES in subjects with type 1 diabetes as compared to normal, non-diabetic controls. This distinct profile of cytokine and chemokine expression can be used to detect or diagnose type 1 diabetes or detect an increased likelihood of developing diabetes.

[0031] Accordingly, in one embodiment the invention includes methods of detecting or diagnosing type 1 diabetes in a subject or detecting an increased likelihood of developing type 1 diabetes in a subject comprising: (a) determining the expression of one or more cytokine or chemokine from a test sample from a subject to generate a subject expression profile, wherein the cytokine comprises IL-8, IL-1 alpha or TNF-alpha and wherein the chemokine comprises MIP-1 alpha, MIP-1 beta, RANTES or MIG; and (b) comparing the subject expression profile to a control, wherein a difference in the subject expression profile as compared to the control is indicative of type 1 diabetes or an increased likelihood of developing type 1 diabetes in the subject.

[0032] The phrase "detecting or diagnosing type 1 diabetes" refers to a method or process of determining if a subject has or does not have type 1 diabetes, or the severity of type 1 diabetes. In addition, the invention can be used to detect or monitor the appearance and progression of type 1 diabetes in a subject.

[0033] As used herein, "type 1 diabetes" optionally refers to a subject with a fasting blood glucose level over 7.0 mmol/L, or a random (anytime of day) sugar that is greater than 11.1 mmol/L caused by a lack of insulin.

[0034] As used herein, a subject with an "increased likelihood of developing type 1 diabetes" refers to a subject who does not have a fasting blood glucose level over 7.0 mmol/L, or a random (anytime of day) sugar that is greater than 11.1 mmol/L caused by a lack of insulin but who has a condition involving an abnormal immune response against beta cells of the pancreas that damages and/or kills the beta cells so that they do not produce insulin. The condition is optionally a genetic autoimmune disease. In these subjects, the beta cell population is typically minimally depleted and still capable of producing physiological levels of insulin to maintain glucose homeostasis. A subject with an increased likelihood of developing type 1 diabetes exhibits a difference in the cytokine or chemokine expression profile compared to controls. In one embodiment, a subject with an increased likelihood of developing type 1 diabetes optionally has beta cell population that is depleted less than 10%, less than 30 %, less than 50%, less than 70% or less than 80% compared to control subjects without type 1 diabetes. In a further aspect, a subject with an increased likelihood of developing diabetes may exhibit auto-immunity that leads to progressive beta-cell destruction. One autoantibody found in people with an increased likelihood of developing diabetes is the islet cell antibody. Other antibodies include the GAD (or 64-K) antibody and the ICA 512 antibody.

[0035] The term "subject" as used herein refers to any member of the animal kingdom, preferably a mammal, such as a human being.

[0036] The term "control" as used herein refers to a sample from an individual or a group of individuals who are known as not having type 1 diabetes (non-diabetic, negative control) or who are known as having type 1 diabetes (type 1 diabetic, positive control). The term also includes pre- determined standardized results.

[0037] The term "sample" refers to any fluid from an individual which can be assayed for cytokine and/or chemokine expression. In one embodiment, the sample is serum or plasma. In another embodiment, the sample is plasma. In another embodiment, the sample is culture supernatant. In a further embodiment, the sample is dendritic cell sample, such as a dendritic cell culture supernatant.

[0038] The term "expression profile" as used herein refers to determining the expression of at least one cytokine or chemokine from the group consisting of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha, MIP-1 beta, RANTES and MIG. In one embodiment, the expression profile refers to determining the expression level of two or more cytokines or chemokines from the group consisting of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha, MIP-1 beta, RANTES and MIG. In another embodiment, the expression profile refers to determining the expression level of three or more, four or more, five or more, six or more or all 7 cytokines or chemokines from the group consisting of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha, MIP-1 beta, RANTES and MIG. In a preferred embodiment, the expression profile refers to determining the expression of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha and MIP-1 beta.

[0039] The term "difference in the subject expression profile" refers to comparing the cytokine and/or chemokine expression profile from a subject to an expression profile from a control and determining if and how the subject's profile varies compared to the control. In one embodiment, the control is a non-diabetic control and the IL-8 levels are lower compared to the control, which indicates that the subject has type 1 diabetes an increased likelihood of developing diabetes. In another embodiment, the control is a non-diabetic control and the IL-1 alpha levels are lower compared to the control, which

indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In another embodiment the control is a non- diabetic control and the TNF-alpha levels are lower compared to the control, which indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In another embodiment the control is a non- diabetic control and the MIP-1 alpha levels are lower compared to the control, which indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In another embodiment, the control is a non- diabetic control and the MIP-1 beta levels are lower compared to the control, which indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In a further embodiment, the control is a non- diabetic control and the IL-8 levels are lower, the IL-1 alpha levels are lower the TNF-alpha levels are lower, the MIP-1 alpha levels are lower and the MIP- 1 beta levels are lower compared to the control which indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. Optionally, the aforementioned levels are respectively at least: 25%, 40%, 50%, 60%, 70% or 80% lower than the control. In other embodiments, multivariable methods are used to perform a statistical comparison between expression profiles comprising more than 1 cytokine or chemokine expression level wherein a significant difference between the expression levels indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes.

[0040] In another embodiment, the control is a non-diabetic control and the IL-8 levels are lower, IL-1 alpha levels are lower, TNF-alpha levels are lower, MIP-1 alpha levels are lower, MIP-1 beta levels are lower, MIG levels are lower and/or RANTES levels are higher in the subject as compared to the non-diabetic control, which indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes.

[0041] In one embodiment, the differential expression can be compared using the ratio of the level of expression of a given cytokine or chemokine as compared with the expression level of the given cytokine or chemokine of a

control, wherein the ratio is not equal to 1.0. For example, a ratio of greater than 1 , 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 1 , 0.8, 0.6, 0.4, 0.2, 0.1 , 0.05, 0.001 or less.

[0042] The term "determining the expression of one or more cytokine or chemokine" as used herein refers to measuring or detecting the expression of one or more cytokine or chemokine in a sample. A person skilled in the art will appreciate that a number of methods can be used to measure or detect cytokine or chemokine expression in a sample. These methods include both protein based and nucleic acid based protocols. In some embodiments, plasma or serum cytokine levels are standardized to total leukocyte counts.

[0043] In one embodiment, protein based protocols are used. A person skilled in the art will appreciate that a number of methods can be used to determine the amount of the protein of interest, namely a cytokine or chemokine, including immunoassays such as Western blots, ELISA, immunoprecipitation followed by SDS-PAGE immunocytochemistry. In addition protein arrays, including microarrays, can be used. Protein based protocols may use agents that bind to the protein of interest, namely a cytokine or a chemokine. In one embodiment the agents are antibodies or antibody fragments. [0044] The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies,

bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0045] Antibodies having specificity for a specific protein, such as a cytokine or chemokine, may be prepared by conventional methods. A mammal, (e.g. a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera. [0046] To produce monoclonal antibodies, antibody producing cells

(lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g. the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4:72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Methods Enzymol, 121 :140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246:1275 (1989)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated.

[0047] The invention also contemplates the use of "peptide mimetics" for generating agents that bind to the proteins of interest, namely cytokines and chemokines. Peptide mimetics are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann.

Reports Med. Chem. 24:243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of the isolated proteins of the invention, such as its ability to bind to the proteins of interest. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries.

[0048] Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.

[0049] In one embodiment of the invention, the agents, such as antibodies or antibody fragments, that binds to the protein of interest, such as chemokines or cytokines, are labeled with a detectable marker. [0050] The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²³ I, ¹²⁵ I or ¹³¹ I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion.

[0051] In another embodiment, the detectable signal is detectable indirectly. For example, a labeled secondary antibody can be used to detect the protein of interest.

[0052] In one embodiment of the invention, cytokine and/or chemokine expression is determined using multiplexing technology. This technology has the advantage of quantifying multiple cytokines simultaneously in one sample.

The advantages of this method include low sample volume, cost effectiveness and high throughput screening. Antibody-based multiplexing kits are available from Linco (Millipore Corporation, MA), Bio-Rad Laboratories (Hercules, CA), Biosource (Montreal, Canada), and R&D Systems (Minneapolis, MN).

[0053] In one embodiment of the invention, the test sample is selected from the group consisting of whole blood, plasma and serum. In a further embodiment, the test sample is plasma or serum.

[0054] Nucleic acid based protocols use agents that detect nucleic acid expression products that encode the protein of interest, namely a cytokine or a chemokine. These include RNA transcripts transcribed from genes encoding a cytokine or chemokine.

[0055] A person skilled in the art will appreciate that a number of methods can be used to measure or detect RNA transcripts within a sample, including hybridization or amplification assays. Such assays include arrays (including microarrays), RT-PCR (including quantitative RT-PCR), nuclease protection assays and northern blots. Thus, detection agents include probes and primers specific for the protein of interest.

[0056] The term "primer" as used herein refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis of when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to

prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.

[0057] The term "probe" as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to an RNA product of a gene encoding the protein of interest or a nucleic acid sequence complementary to the RNA product of a gene encoding the protein of interest. The length of probe depends on the hybridize conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length. [0058] The term "hybridize" refers to the sequence specific non- covalent binding interaction with a complementary nucleic acid. One aspect of the invention provides an isolated nucleotide sequence, which hybridizes to a RNA product of a gene encoding a protein of interest or a nucleic acid sequence which is complementary to an RNA product of a gene encoding a protein of interest. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50 ⁰ C may be employed.

[0059] The stringency may be selected based on the conditions used in the wash step. By way of example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50 ⁰ C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C.

[0060] By "at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5 ⁰ C - 16.6 (Log10 [Na+]) + 0.41 (%(G+C) - 600/I), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1 °C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's solution/1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC/0.1% SDS at 60 ⁰ C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42 ⁰ C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6 and in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol.3. [0061] In one embodiment of the invention, the agents that detect the nucleic acid expression products are labeled with a detectable marker. Detection can be direct or indirect.

[0062] Any of the methods of the invention to diagnose or detect type 1 diabetes can be used in addition or in combination with traditional diagnostic techniques for type 1 diabetes.

[0063] The invention also includes kits for diagnosing or detecting type 1 diabetes comprising a detection agent for one or more of the following cytokines or chemokines IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha, MIP-1 beta, RANTES and MIG, and instructions for the use thereof.

[0064] The detection agent in the kit can be for detecting the protein of interest (e.g. antibodies) or can be for detecting nucleic acid expression products encoding the proteins of interest (e.g. probes or primers).

[0065] The inventors also found a distinct pattern of cytokine and chemokine expression by dendritic cells from subjects with type 1 diabetes as compared to normal, non-diabetic controls. Specifically, the inventors found a significantly higher level of the chemokine RANTES in subjects with type 1 diabetes as compared to normal, non-diabetic controls. The inventors also found lower production of IL-6, IL-8, IL-1 alpha, IL-12p70, TNF-alpha, MIP-1 alpha, and MIP-1 beta and increased production of RANTES and MCP-1 by blood dendritic cells in subjects with type 1 diabetes as compared to normal, non-diabetic controls. This distinct profile of cytokine and chemokine expression can be used to detect or diagnose type 1 diabetes or an increased likelihood of developing diabetes.

[0066] Accordingly, in one embodiment the invention includes methods of detecting or diagnosing type 1 diabetes in a subject or detecting an increased likelihood of developing type 1 diabetes in a subject comprising: (a) determining the expression of one or more cytokine or chemokine secreted by blood dendritic cells from a subject to generate a subject expression profile, wherein the cytokine comprises IL-6, IL-8, IL-1 alpha, IL-12p70, or TNF-alpha and wherein the chemokine comprises MIP-1 alpha, MIP-1 beta, RANTES or MCP-1 ; and (b) comparing the subject expression profile to a control, wherein a difference in the subject expression profile as compared to the

control is indicative of type 1 diabetes or an increased likelihood of developing type 1 diabetes in the subject.

[0067] In one embodiment, the blood dendritic cells are isolated from the blood of a subject or control. Dendritic cells may be isolated by magnetic bead separation or by cell sorting using flow cytometry or by other methods known in the art. In a further embodiment, the isolated blood dendritic cells are grown in culture. In one embodiment, the blood dendritic cells are cultured in plasma with CD40 ligand. In other embodiments, dendritic cells are activated by reagents including cross-linking surface antigens (i.e. costimulator molecules like CD40, CD80, CD86; cytokine receptors; toll-like receptors), antigens, and mitogens. In one embodiment, the expression of one or more cytokine or chemokine is determined by assaying for one or more cytokine or chemokine in the dendritic cell culture supernatant.

[0068] The term "subject expression profile" refers to determining the expression of at least one cytokine and/or chemokine from the group consisting of IL-6, IL-8, IL-1 alpha, IL-12p70, TNF-alpha, MIP-1 alpha, MIP-1 beta, RANTES and MCP-1. In one embodiment, the expression profile refers to determining the expression of two or more cytokines and/or chemokines from the group consisting of IL-6, IL-8, IL-1 alpha, IL-12p70, TNF-alpha, MIP- 1 alpha, MIP-1 beta, RANTES and MCP-1. In another embodiment, the expression profile refers to determining the expression of three or more, four or more, five or more, six or more, seven or more, eight or more, or all 9 cytokines and/or chemokines from the group consisting of IL-6, IL-8, IL-1 alpha, IL-12p70, TNF-alpha, MIP-1 alpha, MIP-1 beta, RANTES and MCP-1. [0069] In one embodiment, the control is a non-diabetic control and the

RANTES levels are higher as compared to the non-diabetic control, which indicates that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes. In another embodiment, the IL-6, IL-8, MIP-1 - alpha, MIP-1 -beta are lower and the RANTES levels are higher compared to the control indicating that the subject has type 1 diabetes or an increased likelihood of developing type 1 diabetes.

[0069] In another embodiment, the IL-6 levels are lower, IL-8 levels are lower, IL-1 alpha levels are lower, IL-12p70 levels are lower, TNF-alpha levels are lower, MIP-1 alpha levels are lower, MIP-1 beta levels are lower, RANTES levels are higher and/or MCP-1 levels are higher as compared to the non- diabetic control, which indicates that the subject has type 1 diabetes.

[0070] The invention also includes kits for diagnosing or detecting type

1 diabetes comprising a detection agent for one or more of the following cytokines or chemokines IL-6, IL-8, IL-1 alpha, IL-12p70, TNF-alpha, MIP-1 alpha, MIP-1 beta, RANTES and MCP-1 , and instructions for the use thereof, [0071] The above disclosure generally describes the present invention.

A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0072] In understanding the scope of the present disclosure, the term

"comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. The following non- limiting examples are illustrative of the present invention:

Examples

Example 1 : Plasma Cytokine/Chemokine Levels in established Type 1 Diabetes Mellitus (T1DM) vs. Normal Controls

[0073] SUBJECTS. 25 subjects were recruited from a weekly endocrinology diabetes clinic and separated into two groups: 13 T1 DM- affected individuals and 12 normal healthy subjects. An absolute leukocyte count (LKC) was determined at the time of blood collection for each subject to control for a possible underlying asymptomatic inflammatory response. A glycosylated haemoglobin measurement was also determined on each T1 DM- affected individual as a measure of glycemic control over the past 3 months. All except one subject, a newly-diagnosed T1 DM patient, was on a prescribed regimen of insulin therapy. Furthermore, all donors were asymptomatic for infection and generalized illness at the time of blood collection.

[0074] BLOOD SAMPLE COLLECTION. Venous peripheral blood (~40 mis) was procured from the antecubital region of the forearm by a registered research nurse. An average of 28.6 ± 4.0 mL (n = 25) of blood was collected from each donor into Vacutainer ^® tubes containing sodium heparin

(BD, Franklin Lakes, NJ) and used to isolate DCs for functional studies immediately after collection. An additional 6 mL of peripheral blood was collected in a Vacutainer ^® tube containing K ₂ EDTA (BD) for absolute leukocyte enumeration and HbAi _c determination in the case of diabetic blood samples. Plasma was collected by centrifuging 4 mis of heparinized whole blood at 2000 rpm for 10 minutes, and aliquots immediately frozen at -70°C until ready for cytokine analysis. An appropriate quantity of autologous plasma was added to DC cultures described below.

[0075] CYTOKINE/CHEMOKINE ANALYSIS. The following 15 Th- polarizing cytokines/chemokines were measured: T _H 1 -polarizing: IL-1α, IL-1 β, IFN-α, IL-6, RANTES, MIP-1α, MIP-1β, IFN-γ, IL-12p70, TNF-α, IP-10, MIG and IL-8; T _H 2-polarizing: MCP-1 and IL-10. DC culture supernatants and plasma were thawed on ice and their cytokine/chemokine levels elucidated using multiplexing kits according to manufacturers' instruction (BioSource™

lnternational Inc., Camarillo, CA). A Bio-Plex™ 200 readout System was used (Bio-Rad Laboratories, Hercules, CA), which utilizes Luminex ^® xMAP™ fluorescent bead-based technology (Luminex Corp., Austin, TX). Cytokine levels were automatically calculated from standard curves using Bio-Plex Manager software (v.4.1.1 , Bio-Rad). All values represent mean values of duplicate samples.

[0076] STATISTICAL ANALYSIS. Group comparisons between the normal control and diabetic samples were performed using a non-parametric, unpaired, two-tailed Mann-Whitney test. Median values were compared. In situations requiring comparison to a median value of 0, a Wilcoxon signed- rank test was performed in favour of the Mann-Whitney. The Pearson test was used to determine correlations in cytokine levels. GraphPad Prism software v.5.00 (GraphPad Software Inc. San Diego, CA) was used for all statistical analyses. [0077] Plasma levels of multiple cytokines in human subjects with established type 1 diabetes (T1 DM) were analyzed in comparison with non- diabetic, normal controls. The following cytokine patterns were found to differ between T1 DM subjects (n= 11) and non-diabetic controls (n = 11). All plasma cytokines were standardized to total leukocyte count (LKC). This is important to control for any differences due to the baseline inflammatory state of the individual. All graphs show the value for each individual and the median line.

[0078] As can be seen in Figure 1, significant differences of plasma levels of the pro-inflammatory cytokines IL-8 and IL-1 alpha were found in T1 DM subjects as compared to non-diabetic controls. Specifically, IL-8 (Figure 1A) and IL-1 alpha (Figure 1B) levels were significantly lower in T1DM when standardized to LKC as compared to control subjects.

[0079] Further, as can be seen in Figure 2, the pro-inflammatory cytokine TNF-alpha (Figure 2E) was lower in the plasma of T1 DM subjects as compared to non-diabetic controls.

[0080] The inventors also examined the plasma levels of a number of chemokines in T1 DM subjects. The following plasma levels of chemokines were lower in T1 DM subjects as compared to non-diabetic controls: MIP-1 alpha (Figure 2D), MIP-1 beta (Figure 2A) and MIG (Figure 2C). In contrast, the median plasma level of the chemokine RANTES (Figure 2B) was higher in T1 DM subjects as compared to non-diabetic controls.

[0081] The results of levels and trends of inflammatory cytokines and chemokines in plasma from human subjects with established T1 DM as compared to normal controls are presented in Table 1. Example 2: Secretion Pattern of Inflammatory Cytokines/Chemokines by Dendritic Cells isolated from the peripheral blood of individuals with Type 1 Diabetes vs. Normal Controls

[0082] SUBJECTS. 25 subjects recruited at a weekly endocrinology diabetes clinic were separated into two groups: 13 T1 DM-affected individuals and 12 normal healthy subjects. An absolute leukocyte count (LKC) was determined at the time of blood collection for each subject to control for a possible underlying asymptomatic inflammatory response. A glycosylated haemoglobin measurement was also determined on each T1 DM-affected individual as a measure of glycemic control over the past 3 months. All except one subject, a newly-diagnosed T1 DM patient, was on a prescribed regimen of insulin therapy. Furthermore, all donors attested to being asymptomatic for infection and generalized illness at the time of blood collection.

[0083] BLOOD SAMPLE COLLECTION. Venous peripheral blood (-40 mis) was procured from the antecubital region of the forearm by a registered research nurse. An average of 28.6 ± 4.0 ml_ (n = 25) of blood was collected from each donor into Vacutainer ^® tubes containing sodium heparin

(BD, Franklin Lakes, NJ) and used to isolate DCs for functional studies immediately after collection. An additional 6 mL of peripheral blood was collected in a Vacutainer ^® tube containing K ₂ EDTA (BD) for absolute leukocyte enumeration and HbA-| _C determination in the case of diabetic blood

samples. Plasma was collected by centrifuging 4 mis of heparinized whole blood at 2000 rpm for 10 minutes, and aliquots immediately frozen at -70°C until ready for cytokine analysis. An appropriate quantity of autologous plasma was added to DC cultures described below. [0084] ISOLATION OF DC. Peripheral blood mononuclear cells

(PBMCs) were isolated from whole blood by standard Ficoll-Paque density centrifugation (1.077 g/cm ³ ; Amersham Biosciences; Uppsala, Sweden). Briefly, an equal volume of mixed whole blood and phosphate-buffered saline (PBS) was underlain with Ficoll, centrifugated at 1800 rpm for 25 minutes, and the PBMCs collected. PBMCs were washed twice with 1X PBS and centrifugation at 1500 rpm for 10 minutes. Specific purification of mDC1 , mDC2 and pDC from PBMCs was performed using the magnetic antibody cell sorting (MACS ^® ) Human Blood Dendritic Cell Isolation Kit Il (Miltenyi Biotec, Auburn, CA) according to manufacturers instructions. DC viability and enumeration was assessed using trypan blue exclusion.

[0085] ASSESSING DC PURITY. DC purity was assessed using 3- color flow cytometry. Briefly, DC isolates were labelled with the combined three flurochrome-conjugated monoclonal antibodies HLA-DR-PerCP, CD3- FITC, CD14-FITC and CD19-FITC (BD) for 20 minutes on ice, washed twice with PBS, fixed in 1 % formaldehyde, then immediately read on a FACS Calibur flow cytometer (BD) located at the London Regional Flow Cytometry Facility, Robarts Research Institute, London, Ontario. Antigen expression was analyzed using FlowJo v.7.2 software (TreeStar Inc., Ashland, OR). DC were defined as HLA-DR+ cells lacking CD3, CD14, and CD19 expression. The inventors isolated DC from two independent, random normal subjects as representative. DC purity was > 98% in both subjects.

[0086] DC CULTURE. DCs were cultured in 2 μg/mL CD40 ligand

(R&D Systems, Inc., Minneapolis, MN), which stimulates both mDC and pDC subsets. DCs were cultured at a concentration of 10 ⁶ viable DC/mL in X-Vivo plasma-free media (BioWhitaker, Walkersville, ML) supplemented with 10% autologous plasma. After a 24 hour incubation period at 37 ⁰ C and 5% CO ₂ ,

DCs were pelleted at 2000 rpm for 10 minutes and the cytokine-containing culture supernatant collected and immediately frozen at -70 ⁰ C until ready for cytokine analysis.

[0087] CYTOKINE/CHEMOKINE ANALYSIS. The following 15 Th- polarizing cytokines/chemokines were measured: TH1 -polarizing: IL-1α, IL-1β, IFN-Ci, IL-6, RANTES, MIP-1α, MIP-1β, IFN-γ, IL-12p70, TNF-α, IP-10, MIG and IL-8; T _H 2-polarizing: MCP-1 and IL-10. DC culture supernatants and plasma were thawed on ice and their cytokine/chemokine levels elucidated using multiplexing kits according to manufacturers' instruction (BioSource™ International Inc., Camarillo, CA). A Bio-Plex™ 200 readout System was used (Bio-Rad Laboratories, Hercules, CA), which utilizes Luminex ^® xMAP™ fluorescent bead-based technology (Luminex Corp., Austin, TX). Cytokine levels were automatically calculated from standard curves using Bio-Plex Manager software (v.4.1.1 , Bio-Rad). All values represent mean values of duplicate samples.

[0088] STATISTICAL ANALYSIS. Group comparisons between the normal control and diabetic samples were performed using a non-parametric, unpaired, two-tailed Mann-Whitney test. Because of the extensive range of cytokine concentrations normally found within human plasma, median values were compared rather than the sample mean. In situations requiring comparison to a median value of 0, a Wilcoxon signed-rank test was performed in favour of the Mann-Whitney. The Pearson test was used to determine correlations in cytokine levels. GraphPad Prism software v.5.00 (GraphPad Software Inc. San Diego, CA) was used for all statistical analyses. [0089] Dendritic cells (DC) (> 98% purity) were isolated from peripheral blood of subjects with established T1DM (n = 9) vs. non-diabetic, normal controls (n = 11). Isolated DC were cultured at 10 ⁶ DC/ml and stimulated using CD40 ligation. After 24 hours, culture supernatants were collected, frozen at -7O ⁰ C until assayed for the simultaneous presence of multiple inflammatory cytokines/chemokines.

[0090] As can be seen in Figure 3, a significant difference was seen in

DC secretion of the chemokine RANTES in T1 DM subjects as compared to normal controls. Specifically, the DC secretion of the chemokine RANTES was significantly increased in T1 DM subjects as compared to normal controls. [0091] Other cytokines were examined and the results are shown in

Figure 4. In particular, reduced DC secretion of the pro-inflammatory cytokines IL-6 (Figure 4A), IL-8 (Figure 4B), IL-1 alpha (Figure 4C), TNF- alpha (Figure 4E), and IL-12p70 (Figure 4D) was observed in T1 DM.

[0092] In addition, a number of chemokines were examined and the results are shown in Figure 5. Specifically, DC secretion of the chemokines MIP- 1 alpha (Figure 5A) and MIP- 1 beta (Figure 5B) appeared reduced in T1 DM. While, DC secretion of the chemokine MCP-1 (Figure 5C) appeared enhanced in T1 DM.

[0093] The results of levels and trends of inflammatory cytokines and chemokines secreted by blood dendritic cells from human subjects with established T1 DM as compared to normal controls are presented in Table 2.

Example 3: Serum IL-8 Levels in Age and Sex-matched Subjects with Type 1 Diabetes Mellitus (T1DM) vs. Normal Controls.

Figure 6 shows results from an analysis of IL-8 levels in serum from a separate group of 13 age and sex-matched subjects with type 1 diabetes and non-diabetic controls. Blood samples were collected and prepared, and IL-8 levels were determined as described in Example 1 except that samples of serum were analyzed instead of plasma. IL-8 levels were significantly lower in subjects with type 1 diabetes as compared to control subjects (p = 0.026). Example 4: Classifiers for Type 1 Diabetes

[0094] The inventors have found a distinct pattern of cytokine and chemokine expression in subjects with type 1 diabetes as compared to normal, non-diabetic controls. In one embodiment, the invention includes the use of different classification schemes or statistical models in order to determine whether a subject is identified as having type 1 diabetes or has an

increased likelihood of developing type 1 diabetes based on the chemokine or cytokine expression profiles of the present invention.

[0095] One classification scheme involves testing for a statistically significant difference between the expression levels of individual cytokines or chemokines. For example, a statistically significant difference in the expression of RANTES in blood dendritic cells between a test subject and a non-diabetic control is indicative of diabetes or an increased likelihood of developing diabetes.

[0096] The invention also includes the use of expression profiles that comprise multiple cytokines and/or chemokines. Mathematical models that permit for the use of multiple variables such as regression models, logistic regression models, machine learning, neural networks, principal component analysis combinations, or clustering analysis are all contemplated by the inventors. [0097] Accordingly, one classification scheme of the present invention involves an plasma or serum expression profile comprising expression levels of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha and MIP-1 beta wherein a statistically significant decrease in the expression profile of the test subject compared to a control is indicative of type 1 diabetes or an increased likelihood of developing type 1 diabetes.

[0098] Classification schemes based on the expression profiles disclosed in the present invention can be assessed using techniques known in the art. For example, different classification schemes can be compared using Receiver Operating Characteristic (ROC) curves. A ROC curve is a graphical plot of the sensitivity vs. (1 - specificity) for a binary classifier scheme as its discrimination threshold is varied. The area under the ROC curve (ROC AUC) is a summary statistic which indicates the performance of the classifier scheme. Preferred classification schemes based on the expression profiles disclosed in the present invention include those with an ROC AUC of greater than 0.6, 0.7, 0.8 or 0.9.

Example 5: Threshold Classification Schemes for Type 1 Diabetes

[0099] Other classification schemes involve the use of specific thresholds for each of the cytokine and/or chemokine expression levels of the present invention. In one embodiment, the median values of protein expression found in Tables 1 and 2 can be used to set threshold levels for assessing whether a test subject has type 1 diabetes. For example, referring to Table 1 , a test subject could be classified as having type 1 diabetes if the test sample exhibited IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha, MIP-1 beta and MIG levels less than the indicated median for the normal group and levels of RANTES above the median for the normal group.

[00100] Similarly, referring to Table 2 a test subject could be classified as having type 1 diabetes if the observed secretion of cytokines by dendritic cells in a test subject was above the median for the normal group for RANTES and MCP-1 , and below the median for the normal group for IL-6, IL-8, IL-1 alpha, IL-12p70, TNF-alpha, MIP-1 alpha and MIP-1 beta.

Example 6: Cytokine and Chemokine Expression Profile Levels Indicate an Increased Likelihood of Developing Type 1 Diabetes.

[00101] Traditionally, the diagnosis of type 1 diabetes occurs only when an individual appears in the clinic with symptoms of hyperglycemia. At this stage, it is believed that 80% to 90% of the insulin-producing pancreatic beta cells are destroyed. Due to the severity of this destruction, reversal of the disease is a tremendous feat and at present is unfeasible. Accordingly, much research is aimed at replacing (via transplantation) or regenerating (via stem cell progenitors) large numbers of new beta cells to rapidly return normal insulin secretion. It is known that T cells are responsible for destruction of the beta cells during type 1 diabetes pathogenesis. Dendritic cells are critical for T cell activation through cell-to-cell contact and secretion of cytokines and chemokines. Accordingly, the observed expression profiles of cytokines and chemokines play a role in the pathogenesis of type 1 diabetes prior to the onset of symptoms such as hyperglycemia and permit for detecting an increased likelihood of a subject developing type 1 diabetes. In one

embodiment, the invention therefore includes methods to identify subjects with an increased likelihood of developing type 1 Diabetes at this early, asymptomatic stage when the beta cell population is minimally depleted and still capable of producing physiological levels of insulin to maintain glucose homeostasis.

[00102] While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00103] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

TABLE 1

1 ND = no difference between T1 DM and normal controls

2 nd = not detected

TABLE 2

1 ND = no difference between T1 DM and normal controls

2 nd = not detected