WO2009015472A1 - Procédés pour diagnostiquer un diabète de type 1 - Google Patents

Procédés pour diagnostiquer un diabète de type 1 Download PDF

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Publication number
WO2009015472A1
WO2009015472A1 PCT/CA2008/001383 CA2008001383W WO2009015472A1 WO 2009015472 A1 WO2009015472 A1 WO 2009015472A1 CA 2008001383 W CA2008001383 W CA 2008001383W WO 2009015472 A1 WO2009015472 A1 WO 2009015472A1
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WIPO (PCT)
Prior art keywords
diabetes
subject
alpha
type
expression
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PCT/CA2008/001383
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English (en)
Inventor
Kelly Summers
Richard Nadeau
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London Health Sciences Centre Research Inc.
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Publication of WO2009015472A1 publication Critical patent/WO2009015472A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • 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.
  • 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).
  • 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.
  • dendritic cells are critical for activating T cells particularly during times of infection whereby T cells are required to clear disease-causing pathogens.
  • 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".
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject is human.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • test sample is selected from the group consisting of whole blood, plasma and serum. In one embodiment the test sample is plasma or serum.
  • the expression of the cytokine or chemokine is determined by measuring or detecting RNA transcripts.
  • the step of determining the expression comprises RT-PCR or quantitative RT-PCR of the test sample.
  • the step of determining expression comprises microarray analysis of the test sample.
  • the RT-PCR is conducing using multiplexing technology.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject is human.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the dendritic cells are in cell culture.
  • the step of determining the expression comprises the analysis of cell culture media.
  • 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.
  • the expression of the cytokine or chemokine is determined using protein based protocols.
  • the expression of the cytokine or chemokine is determined using nucleic acid based protocols.
  • the invention includes kits for detecting the likelihood of developing type 1 diabetes in a subject.
  • 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).
  • 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).
  • 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).
  • 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).
  • Figure 5 shows the level of the chemokines MIP-1 alpha (A),
  • 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.
  • 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.
  • 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.
  • the invention can be used to detect or monitor the appearance and progression of type 1 diabetes in a subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • control 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.
  • sample refers to any fluid from an individual which can be assayed for cytokine and/or chemokine expression.
  • the sample is serum or plasma.
  • the sample is plasma.
  • the sample is culture supernatant.
  • the sample is dendritic cell sample, such as a dendritic cell culture supernatant.
  • expression profile 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.
  • 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.
  • 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.
  • the expression profile refers to determining the expression of IL-8, IL-1 alpha, TNF-alpha, MIP-1 alpha and MIP-1 beta.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • aforementioned levels are respectively at least: 25%, 40%, 50%, 60%, 70% or 80% lower than the control.
  • 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.
  • 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.
  • 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.
  • determining the expression of one or more cytokine or chemokine refers to measuring or detecting the expression of one or more cytokine or chemokine in a sample.
  • methods include both protein based and nucleic acid based protocols.
  • plasma or serum cytokine levels are standardized to total leukocyte counts.
  • 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.
  • 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.
  • the agents are antibodies or antibody fragments.
  • 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.
  • 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.
  • Antibodies having specificity for a specific protein may be prepared by conventional methods.
  • a mammal e.g. a mouse, hamster, or rabbit
  • 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.
  • 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.
  • antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
  • 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.
  • 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.
  • 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.
  • 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.
  • the label is preferably capable of producing, either directly or indirectly, a detectable signal.
  • 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.
  • the detectable signal is detectable indirectly.
  • a labeled secondary antibody can be used to detect the protein of interest.
  • cytokine and/or chemokine expression is determined using multiplexing technology. This technology has the advantage of quantifying multiple cytokines simultaneously in one sample.
  • 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).
  • 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.
  • 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.
  • RNA transcripts within a sample, including hybridization or amplification assays.
  • assays include arrays (including microarrays), RT-PCR (including quantitative RT-PCR), nuclease protection assays and northern blots.
  • detection agents include probes and primers specific for the protein of interest.
  • primer 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.
  • probe refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50 0 C.
  • the temperature in the wash step can be at high stringency conditions, at about 65°C.
  • 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.
  • 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.
  • 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.
  • 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.
  • the agents that detect the nucleic acid expression products are labeled with a detectable marker. Detection can be direct or indirect.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • cross-linking surface antigens i.e. costimulator molecules like CD40, CD80, CD86; cytokine receptors; toll
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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]
  • 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
  • Example 1 Plasma Cytokine/Chemokine Levels in established Type 1 Diabetes Mellitus (T1DM) vs. Normal Controls
  • T1 DM- affected individuals 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.
  • CYTOKINE/CHEMOKINE ANALYSIS The following 15 Th- polarizing cytokines/chemokines were measured: T H 1 -polarizing: IL-1 ⁇ , IL-1 ⁇ , IFN- ⁇ , IL-6, RANTES, MIP-1 ⁇ , MIP-1 ⁇ , IFN- ⁇ , IL-12p70, TNF- ⁇ , 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 (BioSourceTM lnternational Inc., Camarillo, CA).
  • Bio-PlexTM 200 readout System was used (Bio-Rad Laboratories, Hercules, CA), which utilizes Luminex ® xMAPTM 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.
  • the pro-inflammatory cytokine TNF-alpha (Figure 2E) was lower in the plasma of T1 DM subjects as compared to non-diabetic controls.
  • 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).
  • the median plasma level of the chemokine RANTES ( Figure 2B) was higher in T1 DM subjects as compared to non-diabetic controls.
  • 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
  • T1 DM-affected individuals 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.
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • PBS phosphate-buffered saline
  • mDC1 , mDC2 and pDC from PBMCs were purified 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.
  • 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.
  • DC CULTURE DCs were cultured in 2 ⁇ g/mL CD40 ligand
  • 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.
  • CYTOKINE/CHEMOKINE ANALYSIS The following 15 Th- polarizing cytokines/chemokines were measured: TH1 -polarizing: IL-1 ⁇ , IL-1 ⁇ , IFN-Ci, IL-6, RANTES, MIP-1 ⁇ , MIP-1 ⁇ , IFN- ⁇ , IL-12p70, TNF- ⁇ , 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 (BioSourceTM International Inc., Camarillo, CA).
  • Bio-PlexTM 200 readout System was used (Bio-Rad Laboratories, Hercules, CA), which utilizes Luminex ® xMAPTM 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.
  • DC Dendritic cells
  • Example 3 Serum IL-8 Levels in Age and Sex-matched Subjects with Type 1 Diabetes Mellitus (T1DM) vs. Normal Controls.
  • Example 4 Classifiers for Type 1 Diabetes
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Other classification schemes involve the use of specific thresholds for each of the cytokine and/or chemokine expression levels of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention a pour objet des procédés pour détecter et diagnostiquer un diabète de type 1 à l'aide d'une cytokine et/ou d'un profil d'expression de chimiokine où la cytokine comprend l'IL-8, l'IL-1 alpha, l'IL-6, l'IL-12p70 ou le TNF alpha et où la chimiokine comprend MIP-1 alpha, MIP-1 bêta, RANTES, MIG ou MCP-1. L'invention concerne également des kits en vue d'une utilisation avec les procédés de l'invention.
PCT/CA2008/001383 2007-07-30 2008-07-30 Procédés pour diagnostiquer un diabète de type 1 WO2009015472A1 (fr)

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CN107206051A (zh) * 2014-10-24 2017-09-26 法玛科技顾问股份有限公司 巨噬细胞发炎蛋白‑1β（MIP‑1β）抑制剂用以保护胰脏及防止血糖升高的用途

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2504783C1 (ru) * 2012-12-26 2014-01-20 Екатерина Александровна Репина Способ диагностики аутоиммунных вариантов сахарного диабета
CN107206051A (zh) * 2014-10-24 2017-09-26 法玛科技顾问股份有限公司 巨噬细胞发炎蛋白‑1β（MIP‑1β）抑制剂用以保护胰脏及防止血糖升高的用途

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