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WO2015026893A1 - Biomarqueurs prédictifs pour syndrome métabolique - Google Patents

Biomarqueurs prédictifs pour syndrome métabolique Download PDF

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WO2015026893A1
WO2015026893A1 PCT/US2014/051791 US2014051791W WO2015026893A1 WO 2015026893 A1 WO2015026893 A1 WO 2015026893A1 US 2014051791 W US2014051791 W US 2014051791W WO 2015026893 A1 WO2015026893 A1 WO 2015026893A1
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ucp
fasn
subject
protein
antibodies
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Patrick J. Muraca
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Nuclea Biotechnologies Inc
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Nuclea Biotechnologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • 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
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention provides predictive biomarkers and methods of use for the determination of insulin resistance and sensitivity, in addition to cardiometabolic risk and risk associated with obesity. Methods for the stratification of patients along continuum of susceptibility to cardiometabolic risk are also provided.
  • cardiometabolic risk carries enormous therapeutic and economic implications. Whereas there is general agreement that, at least from an epidemiologic perspective, obesity poses a cardiometabolic risk, it is also true that not all obese individuals develop
  • lipid metabolism is as important, if not more important, to diabetes as is carbohydrate metabolism.
  • the anabolic effects of insulin are not limited to facilitating glucose uptake.
  • insulin is the most lipogenic hormone and exerts important effects on protein metabolism.
  • insulin is an important regulator of fatty acid synthase (FASN), a key enzyme in de novo lipogenesis.
  • FASN fatty acid synthase
  • acetyl CoA carboxylase along with FASN determines the lipogenic flux from malonyl CoA into palmitate.
  • Expression of the FASN gene is primarily regulated by hormonal and nutritional signals, and insulin particularly not only increases the rate of FASN gene transcription in murine cell lines and primary human adipocytes but also increases human FASN gene expression and FASN enzymatic activity.
  • FASN is markedly inactivated under conditions of insulin resistance.
  • a marker related to the de novo lipogenic pathway might provide some insights on the level of impairment of insulin sensitivity and metabolic stress.
  • the levels of FASN including variant forms of FASN, such as single nucleotide polymorphisms may serve as a biomarker for metabolic syndrome or any of the component conditions associated with the syndrome.
  • FASN is a potential marker for metabolic stress
  • FASN an intracellular protein
  • Insulin In addition to components in lipogenesis, such as FASN, Insulin also regulates the expression of other metabolic genes that prevent excessive lipid storage in adipose tissues, i.e. genes for lipid homeostasis. It is suggested that Insuline can induce the genetic expression of UCP-1, uncoupling protein- 1, in brown adipocytes (Valverde AM et al Insulin induced up-regulated uncoupling protein- 1 expression is mediated by insulin receptor substate 1 through the phosphatidylinositol 3 -kinase/ Akt signaling pathway in fetal brown adipocytes. J Biol Chem, 2003, Mar 21; 278, 10221-10231).
  • UCP-l (also called thermogenin), that is exclusively expressed in the mintochondria of brown adipocytes, is a key player in thermogenesis to generate heat by uncoupling oxidative phosphorylation, which uncouples the respiratory chain, allowing for fast substrate oxidation with a low rate of ATP product.
  • UCP-1 is activated in the brown fat cell by fatty acid and inhibited by nucleotides. It is hypothezied that UCP-1 has been related to the development of obesity, diabetes mellitus and other related diseases through the link between the control of body temperature to body weight regulation.
  • the present invention provides predictive biomarkers and methods of use for the determination of insulin resistance and sensitivity, in addition to cardiometabolic risk and risks associated with obesity. Methods for the stratification of patients along continuum of susceptibility to cardiometabolic risk and/or metabolic syndrome are also provided. [0010]
  • the present invention provides a method for predicting the incidence of metabolic syndrome in a subject comprising determining the level of UCP-1 or UCP-1 in combination with FASN in a sample obtained from the subject and stratifying the subject as likely to develop metabolic syndrome based on the level of UCP-1 or UCP-1 in combination with FASN where the detection rate of either UCP-1 or UCP-1 in
  • combination with FASN may be independently 0.90 or greater.
  • the subject may have been diagnosed with type 2 diabetes and may also be resistant to insulin.
  • the protein expression levels may be measured by methods such as, but not limited to,
  • the immunohistochemical assay which may utilize one or more UCP-1 specific antibodies.
  • the UCP-1 specific antibodies may further contain a detectable label.
  • the methods of predicting the incidence of metabolic syndrome may further comprise measuring one or more clinical management parameter such as, but not limited to, blood pressure, body mass index (BMI), levels of insulin, blood sugar, triglycerides, HDL, LDL and C-reactive protein.
  • clinical management parameter such as, but not limited to, blood pressure, body mass index (BMI), levels of insulin, blood sugar, triglycerides, HDL, LDL and C-reactive protein.
  • the present invention provides an immunohistochemical kit or assay to predict or detect metabolic syndrome comprising one or more UCP-1 specific antibodies which may comprise a detectable label.
  • the kit or assay may further comprise a probe targeting the FASN gene or protein and/or a probe targeting the USP2a gene or protein.
  • the present invention provides a method of predicting whether a subject afflicted with metabolic syndrome will develop cardiaovascular diseases, (i.e.heart failure) comprising obtaining a biologic sample from the subject, determining the level of UCP-1 and the level of one or more biomarkers selected from the group consisting of FASN, USP2A, ⁇ 8 ⁇ 1, SOD2, KCNE2 and BNP in said biologic sample and stratifying the subjects as likely to develop heart failure based on the expression level of said one or more biomarkers.
  • the subject has also been diagnosed with type 2 diabetes.
  • the subject is also insulin resistant.
  • protein expression levels are measured.
  • the invention provides a method of predicting the incidence of metabolic syndrome in a subject comprising obtaining a biologic sample from the subject and determining the expression level of one or more biomarkers in the biologic sample selected from the group consisting of FASN, USP2A and UCP-1.
  • the biologic sample obtained is selected from the group consisting of blood, peripheral blood mononuclear cells (PBMC), isolated blood cells, serum and plasma.
  • protein expression levels are measured by immunoassay.
  • the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
  • the expression level of three biomarkers is determined.
  • the invention provides a method of predicting the progression and/or regression of metabolic syndrome in a subject comprising obtaining a biological sample from the subject and determining the expression level of UCP-1, or UCP-1 in combination of FASN, or UCP-1 in combination with FASN and USP2A in the biological sample.
  • the subject may be diagnosed as Type 2 diabetes and may also be insulin resistant.
  • the subject may be under treatment for type 2 diabetes.
  • the protein expression level is determined by immunoassay.
  • the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
  • the invention relates to compositions, methods and assays for detecting, screening for, or diagnosing conditions including, but not limited to metabolic syndrome, heart failure, insulin resistance; staging or stratifying subjects; and determining the progression of, regression of and/or survival from metabolic syndrome.
  • metabolic syndrome refers to a group of risk factors that occur together and increase an individual's risk for coronary artery disease, heart failure (HF) [also referred to herein as congestive heart failure (CHF)], stroke, and Type 2 diabetes.
  • HF heart failure
  • CHF congestive heart failure
  • Metabolic syndrome in Type 2 diabetes is characterized by the presentation of hyperinsulinemia, meaning a fasting insulin in the upper 25% of the diabetic population, e.g., elevated fasting blood glucose.
  • metabolic syndrome presents with central obesity and any two of the following: (1) raised triglycerides (TG) of >150 mg/dL (1.7mmol/L), or specific treatment for increased triglycerides; (2) reduced high density lipoproteins (HDL) of ⁇ 40 mg/dL (1.03 mmol/L) in males ⁇ 50 mg/dL (1.29 mmol/L in females; (3) raised blood pressure (BP) with systolic >130 or diastolic >85 mm Hg or treatment for hypertension and (4) raised fasting plasma glucose (FPG) >100 mg/dL (5.6 mmol/L) or previous diagnosis of type 2 diabetes.
  • TG raised triglycerides
  • HDL high density lipoproteins
  • BP blood pressure
  • FPG fasting plasma glucose
  • Metabolic syndrome may also be defined as presentation of hyperinsulinemia and any two of the following: (1) abdominal obesity (waist/hip ration >0.90 or BMI 30 kg/ m 2 ), (2) dyslipidemia (triglycerides (TG) >1.7 or high density lipoprotein (HDL) ⁇ 0.9 mmol/L) and (3) hypertension (blood pressure (BP) > 140/90 or use of antihypertensive medication).
  • HF heart failure
  • CHF congestive heart failure
  • cardiovascular complications associated with MS including, but not limited to myocardial infarction (heart attacks) and other forms of ischemic heart disease, hypertension, valvular heart disease, and
  • Heart failure can cause a number of symptoms including shortness of breath (typically worse when lying flat, which is called orthopnea), coughing, chronic venous congestion, ankle swelling, and exercise intolerance. Heart failure is often undiagnosed because of a lack of a universally agreed definition and challenges in definitive diagnosis. Treatment commonly consists of lifestyle measures (such as smoking cessation, light exercise including breathing protocols, decreased salt intake and other dietary changes) and medications, and sometimes devices (pacemaker) or even surgery.
  • lifestyle measures such as smoking cessation, light exercise including breathing protocols, decreased salt intake and other dietary changes
  • Class I no limitation is experienced in any activities; there are no symptoms from ordinary activities.
  • Class II slight, mild limitation of activity; the patient is comfortable at rest or with mild exertion.
  • Class III marked limitation of any activity; the patient is comfortable only at rest.
  • Class IV any physical activity brings on discomfort and symptoms occur at rest.
  • the NYHA score documents severity of symptoms, and can be used to assess response to treatment. While its use is widespread, the NYHA score is not very reproducible and doesn't reliably predict the walking distance or exercise tolerance on formal stress testing. Raphael C, Briscoe C, Davies J, et al. (2007) "Limitations of the New York Heart Association functional classification system and self-reported walking distances in chronic heart failure", Heart 93(4): 476-82.
  • Stage A At high risk for developing HF in the future but without structural heart disease or symptoms of HF;
  • Stage B Structural heart disease but without signs or sympoms of HF;
  • Stage C Structural heart disease but with prior or current symptoms of HF;
  • Stage D Refractory HF requiring specialized interventions (hospital-based support, a heart transplant or palliative care).
  • ACC staging system is useful in that Stage A encompasses "pre-heart failure", a stage at which therapeutic intervention can presumably prevent progression to overt symptoms. ACC stage A does not have a corresponding NYHA class. ACC Stage B would correspond to NYHA Class I. ACC Stage C corresponds to NYHA Classes II and III, while ACC Stage D overlaps with NYHA Class IV.
  • Imaging e.g., MRI
  • electrocardiography e.g., an electrocardiogram
  • ECG/EKG may be used to identify arrhythmias, ischemic heart disease, right and left ventricular hypertrophy, and presence of conduction delay or abnormalities); and blood tests.
  • Blood tests routinely performed include electrolytes (sodium, potassium), measures of renal function, liver function tests, thyroid function tests, a complete blood count, and often C-reactive protein (also a diagnostic marker for MS) if infection is suspected.
  • An elevated B-type natriuretic peptide (BNP) is a specific test indicative of heart failure. Additionally, BNP can be used to differentiate between causes of dyspnea due to heart failure from other causes of dyspnea. If myocardial infarction is suspected, various cardiac markers may be used. BNP is a useful indicator for heart failure and left ventricular systolic dysfunction.
  • Prognosis in heart failure can be assessed in multiple ways including clinical prediction rules and cardiopulmonary exercise testing.
  • Clinical prediction rules use a composite of clinical factors such as lab tests and blood pressure to estimate prognosis.
  • the 'EFFECT rule slightly outperformed other rules in stratifying patients and identifying those at low risk of death during hospitalization or within 30 days.
  • Biomarkers that are predictive of the risk of a HF patient advancing to a later stage of disease include, but are not limited to glutathione S-transferase omega- 1 ( ⁇ ), superoxide dismutase 2 (SOD2), potassium voltage-gated channel subfamily E member 2 (KCNE2), fatty acid synthase (FASN),Ubiquitin specific cysteine protease 2A (USP2A), and B-type natriuretic peptide (BNP). Both ⁇ and SOD2 are involved in oxidative stress management while BNP and KCNE2 proteins modulate blood pressure and cardiac contraction respectively.
  • biomarker panels can also be used to identify those individuals at highest risk for diabetes and related metabolic disorders (including cardiovascular disease, e.g. Heart Failure).
  • This study indicates the uncoupling protein UCP-1, as highly associated with insulin resistance in type 2 diabetes and obesity, is a biomarker for metabolic syndrome.
  • these biomarkers may be used to determined the risk of developing MS and/or HF.
  • these biomarkers may be used to diagnose MS and/or HF that the subject is experiencing or has experienced.
  • these biomarkers may be used to monitor the progression of MS and /or HF during medical treatments.
  • combinations of UCP-1 with one or more other biomarkers may be detected in a subject to diagnose, prognose or otherwise evaluate an individual with MS to enhance the diagnostic and/or prognostic values of these biomarkers analyzed seprately.
  • combinations of UCP-1 with HF-related biomarkers as discussed above, but not limited to, may be detected in an individual with MS to determine the risk of HF.
  • the present invention provides compositions, methods, kits and other clinical tools to augment traditional diagnostic, prognostic and/or therapeutic paradigms.
  • Combination approaches using one or more biomarkers in the determination of the value of one or more clinical management parameters also are envisioned.
  • methods of this invention that measure UCP- 1 , FASN and USP2A biomarkers, alone or in combination can provide potentially superior results to diagnostic assays measuring just one of these biomarkers.
  • a dual or triple-biomarker approach would provide even further superiority. Any dual, or triple, biomarker approach (with or without companion testing such as blood pressure, triglycerides, etc) thus reduces the number of patients that are predicted not to benefit from treatment, and thus potentially reduces the number of patients that fail to receive treatment that may extend or improve their life significantly.
  • Clinical Management Parameters addressed by the present invention include, but are not limited to, survival in years, disease related death, early or late response to insulin and resistance, degree of regression, responsiveness to treatment, effectiveness of treatment, the likelihood of progression of a condition on to a more severe disease such as one or more cancers, blood pressure, body mass index (BMI), levels of insulin, blood sugar, triglycerides, HDL, LDL, C-reactive protein, as well as biomarker status such as levels of UCP-1, FASN, USP2A, GSTQl, SOD2, KCNE2, BNP or other genes or a SNP of FASN, GSTQl, SOD2, KCNE2, BNP or USP2A, or any metabolic related gene.
  • BMI body mass index
  • practice of the present invention can result in reduced harms caused by screening (resulting in false positives or false negative) and unnecessary subsequent evaluations and therapy.
  • the invention relates to compositions, methods and assays for detecting, screening for, or diagnosing metabolic syndrome (MS) and heart failure (HF)and determining the progression of, regression of and/or survival from MS and/or HF.
  • MS metabolic syndrome
  • HF heart failure
  • the present invention provides methods, algorithms and other clinical tools to augment traditional diagnostic, prognostic and/or therapeutic paradigms.
  • Clinical management parameters addressed by the present invention include survival in years, disease related death, degree of progression, responsiveness to treatment and effectiveness of treatment (e.g., increased cardiac output). [0038] Believing that UCP-1 expression is a superior predictor of many clinical management parameters important to clinicians treating patients having or suspected of having metabolic syndrome, the present invention invovles the rapid identification of UCP-1 expression in tissues, cells and/or serum.
  • a method generally comprises the following steps: (a) obtaining a biological sample (optimally containing cells or other cell or fluid) from a subject; (b) contacting the sample with a detection agent specific for UCP-1 ; (c) detecting the presence, amount or levels of UCP-1 in (b); and (d) correlating the presence, amount or levels of UCP-1 (alone or in combination with one or more other biomarkers) with the one or more clinical management parameters in order to aid in the prevention, diagnosis or treatment of a disease or condition such as metabolic syndrome.
  • the detection agent may be a nucleic acid probe to UCP-1, protein specific for UCP-1, or an anti-UCP-1 antibody.
  • a method generally comprises the following steps: (a) obtaining a biological sample (optimally containing cells or other cell or fluid) from a subject; (b) contacting the sample with a detection agent specific for UCP-1, together with FASN and/or USP2a, together with one or more of ⁇ , SOD2, KCNE2 and BNP; (c) detecting the presence, amount or levels of the markers in (b); and (d) correlating the presence, amount or levels of the markers in order to aid in the prevention, diagnosis or treatment of a condition such as heart failure (HF).
  • Step (d) may further include correlating the marker levels with one or more clinical management parameters and/or imaging data.
  • Clinical management parameters may include, for example, stress testing, cardiac echocardiogram, and cardiac enzymes in blood.
  • the biological sample may be cells or blood, and preferably is serum or plasma containing cells [including, for example and without limitation peripheral blood mononuclear cells (PBMCs)].
  • PBMCs peripheral blood mononuclear cells
  • the cells also may be obtained from cell cultures such as in ex vivo or in situ methods.
  • the detection agent may a nucleic acid probe specific for one or more of UCP- 1,FASN, USP2a, GSTQl, SOD2, KCNE2 and BNP, or an antibody specific for one or more of UCP-1, FASN, USP2a, GSTQl, SOD2, KCNE2 and BNP.
  • the present invention also provides nucleic acid based probes useful in the detection of the UCP-1 gene or protein in a biological sample.
  • the present invention includes nucleic acid sequences specific for segments of a human UCP-1 gene (NCBI accession No. NM_021833, SEQ ID NO. 1) which are used in methods of detecting UCP-1 specific sequences, in nucleic acids prepared from a biological sample.
  • a nucleic acid molecule with a nucleic acid sequence specific to UCP-1 gene is selected to detect the presence of UCP-1.
  • the invention further includes nucleic acid sequences specific for segments of other genetic markers, such as a human FASN and/or USP2a.
  • the biological sample may be cells or blood, and preferably is serum or plasma containing cells PBMCs. However, the cells also may be obtained from cell cultures such as in ex vivo or in situ methods.
  • genes whose measurement of gene expression, protein levels or variants may have diagnostic, prognostic or therapeutic value, alone or in combination include ⁇ (glutathione-S-transferase omega- 1), SOD2 (superoxide dismutase 2), KCNE2 (potassium voltage-gated channel subfamily E member 2), BNP (B-type natriuretic peptide), the FTO (fat mass and obesity associated) gene, MC4R (melanocortin 4 receptor), TMEM18 (transmembrane protein 18), GNPDA2
  • genes or proteins may be measured in a subject who has received no treatment for metabolic syndrome or those who have received treatment such as with metformin.
  • the biological sample may include, for example, blood, peripheral blood mononuclear cells (PBMC), isolated blood cells, serum, plasma, lymph node, breast or breast cyst, kidney, liver, lung, muscle, stomach or intestinal tissue.
  • PBMC peripheral blood mononuclear cells
  • the invention also includes preferred methods that combine nucleic acid sequences for amplifying and detecting UCP-1 -specific sequences, FASN, USP2a, pAKT, NPY, ⁇ 8 ⁇ 1, SOD2, KCNE2, BNP and/or AMACR sequences, individually or in combination.
  • Preferred probes, primers and promoter-primers of the present invention are used for detecting and quantifying the UCP-1 -specific RNA species including variants, iso forms or SNPs.
  • Other embodiments of the invention include methods for detecting FASN, USP2a, AKT, GSTQl, SOD2, KCNE2, BNP, NPY, and/or AMACR RNA species, individually or in combination with each other or UCP-1 sequences.
  • detection of these markers individually and in combination are clinically important because cells from individual patients may express one or more of the markers, such that detecting one or more of the markers decreases the potential of false negatives during diagnosis that might otherwise result if the presence of only one marker was tested.
  • ISH In situ hybridization
  • FISH fluorescence in situ hybridization
  • the present invention provides methods of detecting target nucleic acids via in situ hybridization and fluorescent in situ hybridization using novel probes.
  • the methods of in situ hybridization were first developed in 1969 and many improvements have been made since.
  • the basic technique utilizes hybridization kinetics for RNA and/or DNA via hydrogen bonding. By labeling sequences of DNA or RNA of sufficient length
  • probes of the present invention may be single or double stranded and may be DNA, RNA, or mixtures of DNA and RNA. They may also constitute any nucleic acid based construct. Detectable labels for the probes of the present invention may be radioactive or non-radioactive and the design and use of such labels is well known in the art.
  • the present invention utilizes anti-UCP-1 antibodies and ELISA assay.
  • the anti-UCP-1 antibodies are used as capture antibodies in sandwich ELISA assay.
  • anti-UCP-1 antibodies for the detection of UCP-1 are used.
  • the anti-UCP-1 antibodies may be commercially available antibodies from Abeam antibodies to UCP-1 (Catolog No. Ab23841)(Abcam,
  • the antibodies used in the present invention for detection or capture of UCP-1 are anti-UCP-1 antibodies that are highly specific for human UCP-1 (NCBI accession No. NP 068605, SEQ ID NO.2).
  • the antibodies may be anti-UCP-1 antibodies which specifcally recognize human UCP-1 : purified polyclonal antibody from EMD Millipore (Catalog No. 4091) to human UCP-1 protein.
  • EMD Millipore Catalog No. 4091
  • ELISA prosedures the pair of antibodies that detects UCP-1 cantains a capture antibody which is goat polyclonal affinity purified anti UCP-1 from MYBiosource (Catolog No.
  • anti-UCP-1 antibodies are human IgG, rat IgG or mouse IgG specifically reactive with a UCP-1 peptide which includes an amino acid sequence: SHLHGIKPRYTGTYN (SEQ ID NO. 3).
  • the anti-UCP-1 antibodies used herein may be used for the prediction of metabolic syndrome, either alone or in combination with mearurements of other biomarkers. Measurements may be made for examples, in tissues, cells, serum or plasma patients.
  • UCP-1 expression levels are used as a predictor of insulin resistance.
  • the expression levels of UCP-1 may be used in combination with FASN and USP2a, described herein to predict insulin resistance.
  • UCP-1 alone or in combination with FASN and USP2a, and/or one or more other biomarkers ⁇ 8 ⁇ 1, SOD2, KCNE2, BNP, NPY described herein may be an accurate predictor of insulin resistance in Type 2 Diabetes.
  • the present invention provides for the use of combinations of predictors or biomarkers which, heretofore, have not been known as significant collective indicator combinations. These combinations may form the basis of methods, assays or kits useful in the clinical management of metabolic syndrome and type 2 diabetes.
  • UCP-1 expression is measured relative to the expression of one or more additional genes and/or at one or more different biopsy sites or at a different blood or serum draw time. Comparisons of gene expression within the site and/or at a different time allow conclusions to be drawn about the status of a site or the subject and whether a condition such as (but not limited to) any of the features of metabolic syndrome require further monitoring or clinical management. These conclusions then allow for improved predictions, such as (but not limited to) progression of insulin sensitivity and resistance and overall health outcomes.
  • One set of genes which are particularly useful in these methods includes UCP-1 combined with one or more of FASN, USP2a, GSTQl , SOD2, KCNE2, BNP, pAKT HBAlc and NPY. Additional patient parameters may also be combined with the gene expression data to improve the predictive power of the method. Such patient parameters include age, gender, race, BMI, weight, height or other clinical management parameter.
  • the present invention further comprises assays for determining the gene and/or protein expression profile in a biological sample, and instructions for using the assay.
  • the assay may be based on detection of nucleic acids (e.g., using nucleic acid probes specific for the nucleic acids of interest) or proteins or peptides (e.g., using nucleic acid probes or antibodies specific for the proteins/peptides of interest).
  • the assay comprises an immunohistochemistry (IHC) test in which tissue samples, preferably arrayed in a tissue microarray (TMA), are contacted with antibodies specific for the proteins/peptides identified as biomarkers where detection is taken as being indicative of a relationship between the detected gene and one or more clinical management parameters.
  • IHC immunohistochemistry
  • the assay comprises an inducible protein (IP) or ELISA test in which biologic samples are contacted with antibodies specific for the proteins/peptides as biomarkers where detection is taken as being indicative of a relationship between the detected gene and one or more clinical management parameters or patient parameters.
  • IP inducible protein
  • ELISA test in which biologic samples are contacted with antibodies specific for the proteins/peptides as biomarkers where detection is taken as being indicative of a relationship between the detected gene and one or more clinical management parameters or patient parameters.
  • biomarker or diagnostic methods described herein as part of treatment and/or monitoring regimens to predict the progression to, or effectiveness of treatment of, a subject with any therapeutic provides an advantage over treatment or monitoring regimens that do not include such a biomarker or diagnostic step, in that only that patient population which needs or derives most benefit from such therapy or monitoring need be treated or monitored, and in particular, patients who are predicted not to need or benefit from treatment (where progression is not predicted) with any therapy need not be treated.
  • the present invention further provides a method for treating a patient who may have metabolic syndrome or heart failure, comprising the step of diagnosing a patient's likely progression to metabolic syndrome or heart failure using one or more biomarkers described herein the present invention to evaluate conditions such as (but not limited to) insulin resistance; and a step of administering the patient an appropriate treatment regimen for any of the component conditions associated with metabolic syndrome or heart failure given the patient's age, or other therapeutically relevant criteria.
  • Proteins in the tissue samples may be analyzed by interrogating the TMAs using protein-specific agents, such as antibodies or nucleic acid probes, such as
  • the tissue arrays used may include tissue selected from, but not limited to, the pancreas, colon, gall bladder, kidney, bladder, adipose tissue and muscle pectoralis.
  • Antibodies are preferred for this purpose due to their specificity and availability.
  • the antibodies may be monoclonal or polyclonal antibodies, antibody fragments, and/or various types of synthetic antibodies, including chimeric antibodies, or fragments thereof.
  • Antibodies are commercially available from a number of sources (e.g., Abeam, Cell Signaling Technology or Santa Cruz Biotechnology), or may be generated using techniques well-known to those skilled in the art.
  • the antibodies typically are equipped with detectable labels, such as enzymes, chromogens or quantum dots, which permit the antibodies to be detected.
  • detectable labels such as enzymes, chromogens or quantum dots, which permit the antibodies to be detected.
  • the antibodies may be conjugated or tagged directly with a detectable label, or indirectly with one member of a binding pair, of which the other member contains a detectable label.
  • Detection systems for use with are described, for example, in the website of Ventana Medical Systems, Inc.
  • Quantum dots are particularly useful as detectable labels. The use of quantum dots is described, for example, in the following references: Jaiswal et al., Nat. BiotechnoL, 21 :47-51 (2003); Chan et al, Curr. Opin. BiotechnoL, 13:40-46 (2002); Chan et al., Science, 281 :435-446 (1998).
  • immunohistochemistry The use of antibodies to identify proteins of interest in the cells of a tissue, referred to as immunohistochemistry (IHC), is well established. See, e.g., Simon et al., BioTechniques, 36(1):98 (2004); Haedicke et al., BioTechniques, 35(1): 164 (2003), which are hereby incorporated by reference.
  • the IHC assay can be automated using commercially available instruments, such as the Benchmark instruments available from Ventana Medical Systems, Inc.
  • the TMAs are contacted with antibodies specific for the proteins encoded by the genes identified in the gene expression study as being
  • the antibodies used to interrogate the TMAs are selected based on the genes having the highest level of differential expression.
  • compositions described herein may be comprised in a kit.
  • Antibodies may be included to provide concentrations of from about 0.1 ⁇ g/mL to about 500 ⁇ g/mL, from about 0.1 ⁇ g/mL to about 50 ⁇ g/mL or from about 1 ⁇ g/mL to about 5 ⁇ g/mL or any value within the stated ranges.
  • the kit may further include reagents or instructions for creating or synthesizing further probes, labels or capture agents.
  • kits of the invention may include components for making a nucleic acid or peptide array including all reagents, buffers and the like and thus, may include, for example, a solid support.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial or similar container.
  • kits of the present invention also will typically include a means for containing the detection reagents, e.g., nucleic acids or proteins or antibodies, and any other reagent containers in close confinement for commercial sale.
  • Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • labeling dyes are provided as a dried power.
  • kits of the invention 10-20 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most those amounts of dried dye are provided in kits of the invention.
  • the dye may then be resuspended in any suitable solvent, such as DMSO.
  • Kits may also include components that preserve or maintain the compositions that protect against their degradation.
  • Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
  • Certain assay methods of the invention comprises contacting a tissue sample from an individual with a group of antibodies specific for some or all of the genes or proteins of a GPEP, and determining the occurrence of up- or down-regulation of these genes or proteins in the sample.
  • TMAs allows numerous samples, including control samples, to be assayed simultaneously.
  • the method preferably also includes detecting and/or quantitating control or "reference proteins”. Detecting and/or quantitating the reference proteins in the samples normalizes the results and thus provides further assurance that the assay is working properly.
  • antibodies specific for one or more of the following reference proteins are included: beta-actin (ACTB), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), beta glucoronidase (GUSB) as positive controls while negative controls include large ribosomal protein (RPLP0) and/or transferrin receptor (TRFC). Beta actin may be used as the positive control for IHC.
  • the present invention further comprises a kit containing reagents for conducting an IHC analysis of tissue samples or cells from individuals, e.g., patients, including antibodies specific for one or more proteins and for any reference proteins.
  • the antibodies are preferably tagged with means for detecting the binding of the antibodies to the proteins of interest, e.g., detectable labels.
  • detectable labels include fluorescent compounds or quantum dots; however other types of detectable labels may be used. Detectable labels for antibodies are commercially available, e.g. from Ventana Medical Systems, Inc.
  • immunohistochemical methods for detecting and quantitating protein expression in tissue samples are well known. Any method that permits the determination of expression of several different proteins can be used. Such methods can be efficiently carried out using automated instruments designed for immunohistochemical (IHC) analysis. Instruments for rapidly performing such assays are commercially available, e.g., from Ventana Molecular Discovery Systems or Lab Vision Corporation. Methods according to the present invention using such instruments are carried out according to the manufacturer's instructions. Protein-specific antibodies for use in such methods or assays are readily available or can be prepared using well-established techniques.
  • IHC immunohistochemical
  • the present invention provides for new assays useful in the diagnosis, prognosis and prediction of metabolic syndrome and/or heart failure and the elucidation of clinical management parameters associated with metabolic syndrome and/or heart failure.
  • the immunoassays of the present invention utilize the anti-UCP-1 polyclonal or monoclonal antibodies described herein to specifically bind to UCP-1 in a biological sample.
  • Antibodies to other biomarkers are also available commercially and may be utilized in immunoassays of other embodiments of the present invention.
  • Any type of immunoassay format may be used, including, without limitation, enzyme immunoassays (EIA, ELISA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), counting immunoassay (CIA), immunohistochemistry (IHC), agglutination, nephelometry, turbidimetry or Western Blot.
  • EIA enzyme immunoassays
  • RIA radioimmunoassay
  • FFIA fluoroimmunoassay
  • CLIA chemiluminescent immunoassay
  • CIA counting immunoassay
  • IHC immunohistochemistry
  • agglutination nephelometry
  • turbidimetry turbidimetry or Western Blot.
  • the preferred assay format for the present invention is the enzyme-linked immunosorbent assay (ELISA) format.
  • ELISA is a highly sensitive technique for detecting and measuring antigens or antibodies in a solution in which the solution is run over a surface to which immobilized antibodies specific to the substance have been attached, and if the substance is present, it will bind to the antibody layer, and its presence is verified and visualized with an application of antibodies that have been tagged or labeled so as to permit detection.
  • ELISAs combine the high specificity of antibodies with the high sensitivity of enzyme assays by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number such as alkaline phosphatase (AP) or horseradish peroxidase (HRP), and are very useful tools both for determining antibody concentrations (antibody titer) in sera as well as for detecting the presence of antigen.
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • ELISAs There are many different types of ELISAs; the most common types include "direct ELISA,” “indirect ELISA,” “sandwich ELISA” and cell-based ELISA (C-ELISA).
  • Performing an ELISA involves at least one antibody with specificity for a particular antigen.
  • the sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich” ELISA).
  • a solid support usually a polystyrene microtiter plate
  • the detection antibody is added, forming a complex with the antigen.
  • the detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation.
  • the plate typically is washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound.
  • the plate is developed by adding an enzymatic substrate tagged with a detectable label to produce a visible signal, which indicates the quantity of antigen in the sample.
  • an antibody (“capture antibody”) is adsorbed or immobilized onto a substrate, such as a microtiter plate.
  • Monoclonal antibodies are preferred as capture antibodies due to their greater specificity, but polyclonal antibodies also may be used.
  • the antibody on the plate will bind the target antigen from the sample, and retain it in the plate.
  • a second antibody (“detection antibody”) or antibody pair is added in the next step, it also binds to the target antigen (already bound to the monoclonal antibody on the plate), thereby forming an antigen 'sandwich' between the two different antibodies.
  • This binding reaction can then be measured by radio-isotopes, as in a radioimmunoassay format (RIA); by enzymes, as in an enzyme immunoassay format (EIA or ELISA); or other detectable label, attached to the detection antibody.
  • the label generates a color signal proportional to the amount of target antigen present in the original sample added to the plate.
  • the degree of color can be detected and measured with the naked eye (as with a home pregnancy test), a scintillation counter (for an RIA), or with a spectrophotometric plate reader (for an EIA or ELISA).
  • Assay Steps can be measured by radio-isotopes, as in a radioimmunoassay format (RIA); by enzymes, as in an enzyme immunoassay format (EIA or ELISA); or other detectable label, attached to the detection antibody.
  • the label generates a color signal proportional to the amount of target antigen present in the original sample added to the plate.
  • Step 1 Capture antibodies are adsorbed onto the well of a plastic microtiter plate (no sample added);
  • Step 2 A test sample (such as human serum) is added to the well of the plate, under conditions sufficient to permit binding of the target antigen to the capture antibody already bound to the plate, thereby retaining the antigen in the well;
  • Step 3 Binding of a detection antibody or antibody pair (with enzyme or other detectable moiety attached) to the target antigen (already bound to the capture antibody on the plate), thereby forming an antigen "sandwich" between the two different antibodies.
  • the detectable label on the detection antibodies will generate a color signal proportional to the amount of target antigen present in the original sample added to the plate.
  • the analyte (rather than an antibody) is coated onto a substrate, such as a microtiter plate, and used to bind antibodies found in a sample.
  • a substrate such as a microtiter plate
  • antibodies IgE for example
  • a species-specific antibody (anti-human IgE for example) labeled with an enzyme such as horse radish peroxidase (HRP) is added next, which, binds to the antibody bound to the antigen on the plate.
  • HRP horse radish peroxidase
  • an immunoassay may be structured in a competitive inhibition format.
  • Competitive inhibition assays are often used to measure small analytes because competitive inhibition assays only require the binding of one antibody rather than two as is used in standard ELISA formats.
  • the sample and conjugated analyte are added in steps similar to a sandwich assay, while in a classic competitive inhibition assay, these reagents are incubated together at the same time.
  • a capture antibody is coated onto a substrate, such as a microtiter plate.
  • a substrate such as a microtiter plate.
  • the capture antibody captures free analyte out of the sample.
  • a detectable label such as an enzyme or enzyme substrate.
  • the labeled analyte also attempts to bind to the capture antibody adsorbed onto the plate, however, the labeled analyte is inhibited from binding to the capture antibody by the presence of previously bound analyte from the sample.
  • the labeled analyte will not be bound by the monoclonal on the plate if the monoclonal has already bound unlabeled analyte from the sample.
  • the amount of unlabeled analyte in the sample is inversely proportional to the signal generated by the labeled analyte. The lower the signal, the more unlabeled analyte there is in the sample.
  • a standard curve can be constructed using serial dilutions of an unlabeled analyte standard. Subsequent sample values can then be read off the standard curve as is done in the sandwich ELISA formats.
  • the classic competitive inhibition assay format requires the simultaneous addition of labeled (conjugated analyte) and unlabeled analyte (from the sample). Both labeled and unlabeled analyte then compete simultaneously for the binding site on the monoclonal capture antibody on the plate. Like the sequential competitive inhibition format, the colored signal is inversely proportional to the concentration of unlabeled target analyte in the sample. Detection of labeled analyte can be read on a microtiter plate reader.
  • immunoassays are also may be configured as rapid tests, such as a home pregnancy test. Like microtiter plate assays, rapid tests use antibodies to react with antigens and can be developed as sandwich formats, competitive inhibition formats, and antigen-down formats. With a rapid test, the antibody and antigen reagents are bound to porous membranes, which react with positive samples while channeling excess fluids to a non-reactive part of the membrane.
  • Rapid immunoassays commonly come in two configurations: a lateral flow test where the sample is simply placed in a well and the results are read immediately; and a flow through system, which requires placing the sample in a well, washing the well, and then finally adding an analyte-detectable label conjugate and the result is read after a few minutes.
  • One sample is tested per strip or cassette. Rapid tests are faster than microtiter plate assays, require little sample processing, are often cheaper, and generate yes/no answers without using an instrument.
  • rapid immunoassays are not as sensitive as plate-based
  • a technique for use in the present invention to detect the amount of a biomarker is the sandwich ELISA, in which highly specific monoclonal antibodies are used to detect sample antigen.
  • the sandwich ELISA method comprises the following general steps:
  • the primary antibody (step 5) is linked to an enzyme; in this embodiment, the use of a secondary antibody conjugated to an enzyme (step 6) is not necessary if the primary antibody is conjugated to an enzyme.
  • a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect.
  • an enzyme-linked antibody that binds the Fc region of other antibodies this same enzyme-linked antibody can be used in a variety of situations.
  • the major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample
  • a solid phase substrate such as a microtiter plate or strip, is treated in order to fix or immobilize a capture antibody to the surface of the substrate.
  • the material of the solid phase is not particularly limited as long as it is a material of a usual solid phase used in immunoassays.
  • Such material examples include polymer materials such as latex, rubber, polyethylene, polypropylene, polystyrene, a styrene -butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, a styrene -methacrylate copolymer, polyglycidyl methacrylate, an acrolein-ethyleneglycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), and silicone; agarose; gelatin; red blood cells; and inorganic materials such as silica gel, glass, inert alumina, and magnetic substances. These materials may be used singly or in combination of two or more thereof.
  • the form of the solid phase is not particularly limited insofar as the solid phase is in the form of a usual solid phase used in immunoassays, for example in the form of a microtiter plate, a test tube, beads, particles, and nanoparticles.
  • the particles include magnetic particles, hydrophobic particles such as polystyrene latex, copolymer latex particles having hydrophilic groups such as an amino group and a carboxyl group on the surfaces of the particles, red blood cells and gelatin particles.
  • the solid phase is preferably a microtiter plate or strip, such as those available from Cell Signaling
  • the capture antibody is one or more monoclonal or polyclonal anti-UCP-1 antibodies described herein. Where microtiter plates or strips are used, the capture antibody is immobilized within the wells. Techniques for coating and/or immobilizing proteins to solid phase substrates are known in the art, and can be achieved, for example, by a physical adsorption method, a covalent bonding method, an ionic bonding method, or a combination thereof. See, e.g., W. Luttmann et al., Immunology, Ch. 4.3.1 (pp. 92-94), Elsevier, Inc. (2006) and the references cited therein.
  • a solid phase to which biotin was bound can be used to fix avidin or streptavidin to the solid phase.
  • the amounts of the capture antibody, the detection antibody and the solid phase to be used can also be suitably established depending on the antigen to be measured, the antibody to be used, and the type of the solid phase or the like. Protocols for coating microtiter plates with capture antibodies, including tools and methods for calculating the quantity of capture antibody, are described for example, on the websites for Immunochemistry Technologies, LLC (Bloomington, MN) and Meso Scale Diagnostics, LLC (Gaithersburg, MD).
  • the detection antibody can be any anti-UCP-1 antibody.
  • Anti-UCP-1 antibodies are commercially available, for example, from Cell Signaling Technologies, Inc., Santa Cruz Biotechnology, EMD Biosciences, and others.
  • the detection antibody may be directly conjugated with a detectable label, or an enzyme. If the detection antibody is not conjugated with a detectable label or an enzyme, then a labeled secondary antibody that specifically binds to the detection antibody is included.
  • detection antibody "pairs" are commercially available, for example, fromNovus
  • detectable label refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • the detectable label can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, chemiluminescent compounds , enzymes, and enzyme co-factors, or any other labels known in the art. See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
  • a detectable label can be attached to the subject antibodies and is selected so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures.
  • Appropriate labels include, without limitation, radionuclides, enzymes ⁇ e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or ⁇ - galactosidase), fluorescent moieties or proteins ⁇ e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Evidot® quantum dots supplied by Evident Technologies, Troy, NY, or QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.).
  • the sandwich immunoassay of the present invention comprises the step of measuring the labeled secondary antibody, which is bound to the detection antibody, after formation of the capture antibody-antigen-detection antibody complex on the solid phase.
  • the method of measuring the labeling substance can be appropriately selected depending on the type of the labeling substance. For example, when the labeling substance is a radioisotope, a method of measuring radioactivity by using a radioisotope
  • the labeling substance is a fluorescent substance
  • a method of measuring fluorescence by using a conventionally known apparatus such as a luminometer can be used.
  • the labeling substance is an enzyme
  • a method of measuring luminescence or coloration by reacting an enzyme substrate with the enzyme can be used.
  • the substrate that can be used for the enzyme includes a conventionally known luminescent substrate, calorimetric substrate, or the like.
  • chemilumigenic substrates such as CDP-star® (4-chloro-3- (methoxyspiro (l,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1.- sup.3.7]decane)-4- yl)disodium phenylphosphate) and CSPD® (3-(4-methoxyspiro(l,2-dioxetane-3,2-(5'- chloro)tricyclo[3.3.1.1.sup.3.7]- decane)-4-yl)disodium phenylphosphate) and colorimetric substrates such as p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl- phosphoric acid (BCIP), 4-nitro blue tetrazolium chloride (NBT), and iodonitro tetrazolium (INT
  • the detectable labels comprise quantum dots (e.g., Evidot® quantum dots supplied by Evident Technologies, Troy, NY, or QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif).
  • quantum dots e.g., Evidot® quantum dots supplied by Evident Technologies, Troy, NY, or QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.
  • Techniques for labeling proteins, including antibodies, with quantum dots are known. See, e.g., Goldman et al, Phys. Stat. Sol., 229(1): 407-414 (2002); Zdobnova et al, J. Biomed. Opt., 14(2):021004 (2009); Lao et al, J ACS, 128(46): 14756-14757 (2006); Mattoussi et al, J ACS,
  • quantum dots e.g., Evidot® quantum dots supplied by Evident Technologies, Troy, NY, or Qd
  • Quantum-dot antibody labeling kits are commercially available, e.g., from Invitrogen (Carlsbad, CA) and Millipore (Billerica, MA).
  • the sandwich immunoassay of the present invention may comprise one or more washing steps. By washing, the unreacted reagents can be removed.
  • a washing substance or buffer is contacted with the wells after each step.
  • the washing substance include 2-[N-morpholino]ethanesulfonate buffer (MES), or phosphate buffered saline (PBS), etc.
  • the pH of the buffer is preferably from about pH 6.0 to about pH 10.0.
  • the buffer may contain a detergent or surfactant, such as Tween 20.
  • the sandwich immunoassay can be carried out under typical conditions for immunoassays.
  • the typical conditions for immunoassays comprise those conditions under which the pH is about 6.0 to 10.0 and the temperature is about 30 to 45°C.
  • the pH can be regulated with a buffer, such as phosphate buffered saline (PBS), a
  • TAA triethanolamine hydrochloride buffer
  • the buffer may contain components used in usual immunoassays, such as a surfactant, a preservative and serum proteins.
  • the time of contacting the respective components in each of the respective steps can be suitably established depending on the antigen to be measured, the antibody to be used, and the type of the solid phase or the like.
  • kits comprising agents, which may include gene-specific or gene-selective probes and/or primers, for quantitating the expression of the disclosed genes for predicting prognostic outcome or response to treatment.
  • agents which may include gene-specific or gene-selective probes and/or primers, for quantitating the expression of the disclosed genes for predicting prognostic outcome or response to treatment.
  • kits may optionally contain reagents for the extraction of R A from patient samples, in particular fixed paraffin- embedded tissue samples and/or reagents for RNA amplification.
  • the kits may optionally comprise the reagent(s) with an identifying description or label or instructions relating to their use in the methods of the present invention.
  • kits may comprise containers (including microtiter plates suitable for use in an automated implementation of the method), each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, pre-fabricated microarrays, buffers, and the like.
  • the methods provided by the present invention may also be automated in whole or in part.
  • the invention further provides kits for performing an immunoassay using the UCP-1 antibodies.
  • All aspects of the present invention may also be practiced such that a limited number of additional genes that are co-expressed with the disclosed genes (e.g., one or more genes UCP-1, FASN, USP2a, GSTQl, SOD2, KCNE2, BNP, HBAIC and NPY), for example as evidenced by high Pearson correlation coefficients, are included in a prognostic or predictive tests in addition to and/or in place of disclosed genes.
  • additional genes e.g., one or more genes UCP-1, FASN, USP2a, GSTQl, SOD2, KCNE2, BNP, HBAIC and NPY
  • genomic is intended to include the entire DNA complement of an organism, including the nuclear DNA component, chromosomal or extrachromosomal DNA, as well as the cytoplasmic domain (e.g., mitochondrial DNA).
  • gene refers to a nucleic acid sequence that comprises control and most often coding sequences necessary for producing a polypeptide or precursor. Genes, however, may not be translated and instead code for regulatory or structural RNA molecules. Genes include any variants or isoforms, especially those comprising even a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • a gene may be derived in whole or in part from any source known to the art, including a plant, a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA.
  • a gene may contain one or more modifications in either the coding or the untranslated regions that could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides.
  • the gene may constitute an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions.
  • gene expression refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide.
  • measurements may be of the nucleic acid product of transcription, e.g., RNA or mR A or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.
  • nucleic acid refers to a molecule comprised of one or more nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both.
  • the term includes monomers and polymers of ribonucleotides and deoxyribonucleotides, with the ribonucleotides and/or deoxyribonucleotides being bound together, in the case of the polymers, via 5' to 3' linkages.
  • the ribonucleotide and deoxyribonucleotide polymers may be single or double-stranded.
  • linkages may include any of the linkages known in the art including, for example, nucleic acids comprising 5' to 3' linkages.
  • the nucleotides may be naturally occurring or may be synthetically produced analogs that are capable of forming base-pair relationships with naturally occurring base pairs.
  • Examples of non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogs, aza and deaza purine analogs, and other heterocyclic base analogs, wherein one or more of the carbon and nitrogen atoms of the pyrimidine rings have been substituted by heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and the like.
  • nucleic acids refers to hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DNA molecule or between an oligonucleotide probe and a target are complementary.
  • an "expression product” is a biomolecule, such as a protein or mRNA, which is produced when a gene in an organism is expressed. An expression product may comprise post-translational modifications. The polypeptide of a gene may be encoded by a full length coding sequence or by any portion of the coding sequence.
  • amino acid and “amino acids” refer to all naturally occurring L- alpha-amino acids.
  • the amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M), asparagines (Asn:N), where the amino acid is listed
  • amino acid sequence variant refers to molecules with some differences in their amino acid sequences as compared to a native sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence.
  • variants will possess at least about 70% homology to a native sequence, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to a native sequence.
  • homology as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • homo logs as it applies to amino acid sequences is meant the corresponding sequence of other species having substantial identity to a second sequence of a second species.
  • Analogs is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain the properties of the parent polypeptide.
  • derivative is used synonymously with the term “variant” and refers to a molecule that has been modified or changed in any way relative to a reference molecule or starting molecule.
  • compositions such as antibodies, which are amino acid based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives.
  • polypeptide based molecules containing substitutions, insertions and/or additions, deletions and covalently modifications.
  • sequence tags or amino acids such as one or more lysines, can be added to the polypeptide sequences of the invention (e.g., at the N-terminal or C-terminal ends).
  • Sequence tags can be used for polypeptide purification or localization. Lysines can be used to increase solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.
  • substitutional variants when referring to proteins are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position.
  • the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • conservative amino acid substitution refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine.
  • substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions.
  • non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • deletional variants when referring to proteins, are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
  • Covalent derivatives when referring to proteins, include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
  • Covalent derivatives specifically include fusion molecules in which proteins of the invention are covalently bonded to a non-proteinaceous polymer.
  • the non- proteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer not otherwise found in nature.
  • hydrophilic polyvinyl polymers fall within the scope of this invention, e.g.
  • polyvinylalcohol and polyvinylpyrrolidone are particularly useful. Particularly useful are polyvinylalkylene ethers such a polyethylene glycol, polypropylene glycol.
  • the proteins may be linked to various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.”Features” when referring to proteins are defined as distinct amino acid sequence-based components of a molecule.
  • Features of the proteins of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
  • surface manifestation refers to a polypeptide based component of a protein appearing on an outermost surface.
  • local conformational shape means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
  • fold means the resultant conformation of an amino acid sequence upon energy minimization.
  • a fold may occur at the secondary or tertiary level of the folding process.
  • secondary level folds include beta sheets and alpha helices.
  • tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
  • turn as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
  • loop refers to a structural feature of a peptide or polypeptide which reverses the direction of the backbone of a peptide or polypeptide and comprises four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).
  • domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
  • sub- domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
  • PBMC peripheral blood mononuclear cell
  • monocytes T-cells
  • B-cells natural killer cells
  • site As used herein when referring to proteins the terms "site” as it pertains to amino acid based embodiments is used synonymous with “amino acid residue” and "amino acid side chain".
  • a site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.
  • terminal or terminus when referring to proteins refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions.
  • the polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus [terminated by an amino acid with a free carboxyl group (COOH)].
  • Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini.
  • the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.
  • any of the features have been identified or defined as a component of a molecule of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would. [0130] Modifications and manipulations can be accomplished by methods known in the art such as site directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
  • a "protein” means a polymer of amino acid residues linked together by peptide bonds.
  • a protein may be naturally occurring, recombinant, or synthetic, or any combination of these.
  • a protein may also comprise a fragment of a naturally occurring protein or peptide.
  • a protein may be a single molecule or may be a multi-molecular complex. The term protein may also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a
  • protein expression refers to the process by which a nucleic acid sequence undergoes translation such that detectable levels of the amino acid sequence or protein are expressed.
  • a "fragment of a protein,” as used herein, refers to a protein that is a portion of another protein.
  • fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
  • a protein fragment comprises at least about six amino acids.
  • the fragment comprises at least about ten amino acids.
  • the protein fragment comprises at least about sixteen amino acids.
  • array refer to any type of regular arrangement of objects usually in rows and columns. As it relates to the study of gene and/or protein expression, arrays refer to an arrangement of probes (often oligonucleotide or protein based) or capture agents anchored to a surface which are used to capture or bind to a target of interest. Targets of interest may be genes, products of gene expression, and the like.
  • the type of probe (nucleic acid or protein) represented on the array is dependent on the intended purpose of the array (e.g., to monitor expression of human genes or proteins).
  • the oligonucleotide- or protein-capture agents on a given array may all belong to the same type, category, or group of genes or proteins.
  • Genes or proteins may be considered to be of the same type if they share some common characteristics such as species of origin (e.g., human, mouse, rat); disease state (e.g., cancer, diabetes); structure or functions (e.g., protein kinases, tumor suppressors); or same biological process (e.g., apoptosis, signal transduction, cell cycle regulation, proliferation, differentiation).
  • species of origin e.g., human, mouse, rat
  • disease state e.g., cancer, diabetes
  • structure or functions e.g., protein kinases, tumor suppressors
  • same biological process e.g., apoptosis, signal transduction, cell cycle regulation, proliferation, differentiation.
  • one array type may be a "cancer array” in which each of the array oligonucleotide- or protein- capture agents correspond to a gene or protein associated with a cancer.
  • An "epithelial array” may be an array of oligonucleotide- or protein-
  • immunohistochemical or as abbreviated “IHC” as used herein refer to the process of detecting antigens (e.g., proteins) in a biologic sample by exploiting the binding properties of antibodies to antigens in said biologic sample.
  • antigens e.g., proteins
  • immunoassay refers to a test that uses the binding of antibodies to antigens to identify and measure certain substances. Immunoassays often are used to diagnose disease, and test results can provide information about a disease that may help in planning treatment. An immunoassay takes advantage of the specific binding of an antibody to its antigen. Monoclonal antibodies are often used as they usually bind only to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules. The antibodies used must have a high affinity for the antigen of interest, because a very high proportion of the antigen must bind to the antibody in order to ensure that the assay has adequate sensitivity.
  • PCR or "RT-PCR”, abbreviations for polymerase chain reaction technologies, as used here refer to techniques for the detection or determination of nucleic acid levels, whether synthetic or expressed.
  • cell type refers to a cell from a given source (e.g., a tissue, organ) or a cell in a given state of differentiation, or a cell associated with a given pathology or genetic makeup.
  • activation refers to any alteration of a signaling pathway or biological response including, for example, increases above basal levels, restoration to basal levels from an inhibited state, and stimulation of the pathway above basal levels.
  • differential expression refers to both quantitative as well as qualitative differences in the temporal and tissue expression patterns of a gene or a protein in diseased tissues or cells versus normal adjacent tissue.
  • a differentially expressed gene may have its expression activated or completely inactivated in normal versus disease conditions, or may be up-regulated (over-expressed) or down-regulated (under-expressed) in a disease condition versus a normal condition.
  • Such a qualitatively regulated gene may exhibit an expression pattern within a given tissue or cell type that is detectable in either control or disease conditions, but is not detectable in both.
  • a gene or protein is differentially expressed when expression of the gene or protein occurs at a higher or lower level in the diseased tissues or cells of a patient relative to the level of its expression in the normal (disease-free) tissues or cells of the patient and/or control tissues or cells.
  • RNA expression pattern which is detectable via the standard techniques of polymerase chain reaction (PCR), reverse transcriptase-(RT) PCR, differential display, and Northern analyses, or any method which is well known to those of skill in the art.
  • PCR polymerase chain reaction
  • RT reverse transcriptase-(RT) PCR
  • differential display or any method which is well known to those of skill in the art.
  • protein expression patterns may be "detected” via standard techniques such as Western blots.
  • complementary refers to the topological compatibility or matching together of the interacting surfaces of a probe molecule and its target.
  • the target and its probe can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.
  • antibody means an immunoglobulin, whether natural or partially or wholly synthetically produced. All derivatives thereof that maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.
  • An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE, etc.
  • antibody fragment refers to any derivative or portion of an antibody that is less than full-length. In one aspect, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability, specifically, as a binding partner. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
  • the antibody fragment may be produced by any means. For example, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, the antibody fragment may be wholly or partially synthetically produced.
  • the antibody fragment may comprise a single chain antibody fragment.
  • the fragment may comprise multiple chains that are linked together, for example, by disulfide linkages.
  • the fragment may also comprise a multimolecular complex.
  • a functional antibody fragment may typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts.
  • polyclonal antibody preparations that typically include different antibodies directed against different determinants
  • each monoclonal antibody is directed against a single determinant on the antigen. This type of antibodies is produced by the daughter cells of a single antibody- producing hybridoma.
  • a monoclonal antibody typically displays a single binding affinity for any epitope with which it immunoreacts.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • Monoclonal antibodies recognize only one type of antigen
  • the monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
  • the preparation of antibodies, whether monoclonal or polyclonal, is know in the art. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A
  • a monoclonal antibody may contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody.
  • Monoclonal antibodies may be obtained by methods known to those skilled in the art. Kohler and Milstein (1975), Nature, 256:495-497; U.S. Pat. No. 4,376,110; Ausubel et al. (1987, 1992), eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, N.Y.; Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; Colligan et al.
  • an "antibody preparation” is meant to embrace any composition in which an antibody may be present, e.g., a serum (antiserum).
  • Antibodies may be labeled with detectable labels by one of skill in the art.
  • the label can be a radioisotope, fluorescent compound, chemiluminescent compound, enzyme, or enzyme co-factor, or any other labels known in the art.
  • the antibody that binds to an entity one wishes to measure is not labeled, but is instead detected by binding of a labeled secondary antibody that specifically binds to the primary antibody.
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above.
  • the antibodies of the invention can be from any animal origin including birds and mammals.
  • the antibodies are of human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken origin.
  • Multispecific antibodies can be specific for different epitopes of a peptide of the present invention, or can be specific for both a peptide of the present invention, and a heterologous epitope, such as a heterologous peptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al, 1991, J.
  • the antibodies may be produced against a peptide containing repeated units of a FASN peptide sequence of the invention, or they may be produced against a peptide containing two or more FASN peptide sequences of the invention, or the combination thereof.
  • primer an oligonucleotide capable of binding to a region of a target nucleic acid or its complement and promoting nucleic acid amplification of the target nucleic acid. In most cases a primer will have a free 3' end which can be extended by a nucleic acid polymerase. All amplification primers include a base sequence capable of hybridizing via complementary base interactions either directly with at least one strand of the target nucleic acid or with a strand that is complementary to the target sequence. Amplification primers serve as substrates for enzymatic activity that produces a longer nucleic acid product.
  • a "target-binding sequence" of an amplification primer is the portion that determines target specificity because that portion is capable of annealing to a target nucleic acid strand or its complementary strand.
  • the complementary target sequence to which the target-binding sequence hybridizes is referred to as a primer-binding sequence.
  • detecting an amplification product is meant any of a variety of methods for determining the presence of an amplified nucleic acid, such as, for example, hybridizing a labeled probe to a portion of the amplified product.
  • a labeled probe is an oligonucleotide that specifically binds to another sequence and contains a detectable group which may be, for example, a fluorescent moiety, a chemiluminescent moiety, a radioisotope, biotin, avidin, enzyme, enzyme substrate, or other reactive group.
  • nucleic acid amplification conditions environmental conditions including salt concentration, temperature, the presence or absence of temperature cycling, the presence of a nucleic acid polymerase, nucleoside triphosphates, and cofactors which are sufficient to permit the production of multiple copies of a target nucleic acid or its complementary strand using a nucleic acid amplification method.
  • nucleic acid amplification methods include thermocycling to alternately denature double-stranded nucleic acids and hybridize primers.
  • Biomarker refers to a substance indicative of a biological state.
  • Biomarkers according to the present invention include any compounds or compositions which are used to identify or signal the presence of one or more members selected from UCP-1. FASN and USP2a , or combinations thereof disclosed herein.
  • an antibody created to bind to any of the proteins disclosed herein may be considered useful as a biomarker, although the antibody itself is a secondary indicator.
  • biological sample refers to a sample obtained from an organism (e.g., a human patient) or from components (e.g., cells) or from body fluids (e.g., blood, serum, sputum, urine, etc) of an organism.
  • the sample may be of any biological tissue, organ, organ system or fluid.
  • the sample may be a "clinical sample” which is a sample derived from a patient.
  • Such samples include, but are not limited to, sputum, blood, serum, blood cells (e.g., white cells), peripheral blood mononuclear cells (PBMCs), amniotic fluid, plasma, semen, bone marrow, and tissue or core, fine or punch needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
  • blood cells e.g., white cells
  • PBMCs peripheral blood mononuclear cells
  • amniotic fluid plasma, semen, bone marrow, and tissue or core
  • fine or punch needle biopsy samples e.g., fine or punch needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
  • Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • a biological sample may also be referred to as a "patient sample.”
  • condition refers to the status of any cell, organ, organ system or organism. Conditions may reflect a disease state or simply the physiologic presentation or situation of an entity. Conditions may be characterized as phenotypic conditions such as the macroscopic presentation of a disease or genotypic conditions such as the underlying gene or protein expression profiles associated with the condition. Conditions may be benign or malignant. [0159] The term “syndrome” refers to a collection of symptoms or conditions characterizing a particular clinical etiology. For example, metabolic syndrome is characterized by a collection of physiologic health parameters.
  • body mass index refers to a number calculated from a subject's weight and height that correlates with the level of body fat of a given subject. This value is obtained from a subject by dividing the weight of the subject in kilograms by (height) 2 in meters. BMI values are interpreted as follows: below 18.5 - underweight; 18.5-24.9 - normal; 25.0-29.9 overweight; 30.0 and above - obese.
  • cell growth is principally associated with growth in cell numbers, which occurs by means of cell reproduction (i.e. proliferation) when the rate of the latter is greater than the rate of cell death (e.g. by apoptosis or necrosis), to produce an increase in the size of a population of cells, although a small component of that growth may in certain circumstances be due also to an increase in cell size or cytoplasmic volume of individual cells.
  • An agent that inhibits cell growth can thus do so by either inhibiting proliferation or stimulating cell death, or both, such that the equilibrium between these two opposing processes is altered.
  • clinical management parameter refers to a metric or variable considered important in the detecting, screening, diagnosing, staging or stratifying patients, or determining the progression of, regression of and/or survival from a disease or condition.
  • clinical management parameters include, but are not limited to survival in years, disease related death, early or late recurrence, degree of regression, metastasis, responsiveness to treatment, effectiveness of treatment, the likelihood of progression of a condition, blood pressure, body mass index (BMI), levels of insulin, blood sugar, triglycerides, HDL, LDL, C-reactive protein, as well as biomarker status such as levels of FASN, USP2A, GSTQ 1 , SOD2, KCNE2, BNP or other gene or a SNP of FASN, GSTQ 1 , SOD2, KCNE2, BNP or USP2A, or any metabolic related gene.
  • endpoint means a final stage or occurrence along a path or progression.
  • insulin resistance refers to a physiological condition wherein cells do not respond or do not properly respond to the biological hormone, insulin. Such responses include, but are not limited to insulin binding, insulin-dependent cell signaling, insulin-dependent gene expression, modulation of sugar uptake, modulation of carbohydrate storage and modulation of lipid metabolism.
  • the term "later stage of HF” refers to a stage of HF that occurs as the condition progresses from a less severe stage to a more severe stage, such as from Stage A to Stage B, Stage C or Stage D, from Stage B to Stage C or Stage D, or from Stage C to Stage D. In some emodiments, the term may be used to refer to either of the two final stages of the condition, Stages C and/or D, in the absence of a reference to a prior stage.
  • the term “early stage heart failure” refers to a stage of heart failure that is less severe than a later stage. In some embodiments, early stage heart failure is Stage A, Stage B or Stage C.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the symptoms, conditions, or underlying causes of metabolic syndrome.
  • treatment refers to the act of treating.
  • a method of treating when applied to, for example, metabolic syndrome refers to a procedure or course of action that is designed to reduce, eliminate or prevent development or progression individual, or to alleviate the symptoms of a metabolic syndrome.
  • a method of treating a disorder does not necessarily mean that the disorder will, in fact, be completely eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a disease or other disorder will, in fact, be alleviated. Often, a method of treating will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an individual, is nevertheless deemed an overall beneficial course of action.
  • predicting means a statement or claim that a particular event will, or is very likely to, occur in the future.
  • prognosing means a statement or claim that a particular biologic event will, or is very likely to, occur in the future.
  • progression or “disease progression” means the advancement or worsening of or toward a disease or condition.
  • regression or “degree of regression” refers to the reversal, either phenotypically or genotypically, of a disease progression. Slowing or stopping of any disease progression may be considered regression.
  • the term "stratifying" as it relates to patients means the parsing of patients into groups of predicted outcomes along a continuum of from a positive outcome (such as disease free) to moderate or good outcomes (such as improved quality of life or increased survival) to poor outcomes (such as terminal prognosis or death).
  • the term "subject” or “patient” refers to any organism to which an embodiment of the invention may be applied, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • terapéuticaally effective agent means a composition that will elicit the biological or medical response of a tissue, organ, system, organism, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • terapéuticaally effective amount or “effective amount” means the amount of the subject compound or combination that will elicit the biological or medical response of a tissue, organ, system, organism, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • correlation refers to a relationship between two or more random variables or observed data values.
  • a correlation may be statistical if, upon analysis by statistical means or tests, the relationship is found to satisfy the threshold of significance of the statistical test used.
  • FASN values can be evaluated for insulin resistance based on the degree of regression since the degree of regression is a representation of insulin resistance.
  • the regression analysis of the FASN data revealed that the relationship between FASN and insulin resistance is highly significant as the p-value for the likelihood ratio test and the Pearson's ChiSquare test is ⁇ 0.0001.
  • the biomarkers HBAlc, UCP-1, FASN, USP2a, BMI and NPY were evaluated alone or in combination by logistic regression to determine the ChiSquare p-value to determine their significance on insulin resistance.
  • the results of the analysis are shown in Table 5.
  • the coefficients of the biomarkers UCP-1, HBAlc, FASN and BMI were evaluated. Table 6 shows the estimated coefficient (Estimate), standard error (Std Error) the lower 95% confidence interval (Lower CL) and upper 95% confidence interval (Upper CL) in LogNormal distribution.
  • the 95% confidence interval for the coefficient of HBAlc includes 0 which is an indication that HBAlc is not significant to insulin resistance prediction.
  • the 95% confidence intervals for UCP-1, FASN and BMI do not include zero so they may be significant.
  • FASN and UCP-1 have a negative coefficient which indicates that an increase in value is a predictor for insulin resistance.
  • BMI has a positive coefficient indicating that longer therapy will have a better response to treatment.
  • R 2 (R-squared) is a measure of the whole model. Its value is between 0 and 1. Higher values mean that the model explains more of the overall variation in the data, p-value is a measure of statistical significance. Typically in the art, a p-value below 0.05 is taken as a significant model or effect.
  • ROC Receiveiver Operator Characteristic
  • a ROC-curve involves the count of true positives by false positives as one accumulates the frequencies across a rank ordering.
  • the ROC curve plots (1 -Specificity) on the X-axis and Sensitivity on the Y-axis. The largest possible are under the ROC-Curve is 1 and indicates perfect separation of true positives and true negatives. A value near 1 is desired if the variable is to be predictive.
  • Sensitivity is the probability that a given X-value (a test or measure) correctly predicts the existence of a condition.
  • the X variables are detailed above and the condition would be the binary variable Survive 5 (survival for at least 5 years or not). Specificity is the probability that a test correctly predicts that a condition does not exist.
  • Blocker Blocking Solution 300 ⁇ /well (Thermo) for 2hr at 23°C with shaking at lOOrpm in Incubating Microplate Shaker (VWR) covered with a plate sealer. Afterwards, plates are washed with 5x PBS-T (300 ⁇ /well) on a plate washer. After washing, the plates are tapped on a kimwipe placed on the bench to remove excess liquid.
  • Standards are prepared in advance and included a 7-point dilution (e.g. in 1% BSA in PBS-T from 500 pg/ml). Once prepared, 100 ⁇ of standards or samples freshly diluted in appropriate buffer (PBS-T, R&D Diluent 7, 18 etc.) are loaded at 23°C on plate shaker with lOOrpm agitation for 2hr while covered with plate sealer. Plates are then washed with 5x PBS-T (300ul/well) on a plate washer.
  • appropriate buffer PBS-T, R&D Diluent 7, 18 etc.
  • the detection antibody (100 ⁇ /well; diluted in buffer to appropriate
  • concentration e.g., in PTS/PBS-T
  • PTS/PBS-T concentration, e.g., in PTS/PBS-T
  • the secondary antibody (100 ⁇ /well of appropriate secondary antibody streptavidin-HRP, 1 :200 dilution in PBS) is incubated at 23 °C on plate shaker with lOOrpm agitation for 20min covered with a plate sealer.
  • anti-species-HRP antibody at 1 : 10,000 in PBS for lhr at 23°C on plate shaker with lOOrpm agitation was used.
  • the plates are then washed with 5x PBS-T (300ul/well) on a plate washer.
  • the signal is amplified by adding 100 ⁇ /well R&D Gloset Substrate, for 10 min at room temperature in a BioTek FL800x plate reader.
  • Substrates which are prepared fresh ahead of time are made by mixing Reagent A (stabilized enhanced luminal) with Reagent B (stabilized hydrogen peroxide) in a 1 :2 ratio.
  • Reagent A stabilized enhanced luminal
  • Reagent B stabilized hydrogen peroxide
  • the signal is measured on a BioTek FL800x fluorometer (0.5s read time) with sensitivity auto-adjusted to the highest point on a standard curve and set to a reading of 100,000.
  • ELISA Sandwich assays useful in the present invention include those as described in PCT Publication PCT/US2010/046773 published March 17, 2011, the contents of which are incorporated here by reference in its entirety.
  • FFPE pretreatment is to prepare formalin fixed paraffin-embedded (FFPE) tissue sections fixed on positively charged slides for use in fluorescence in situ hybridization (FISH) with CEP and LSI DNA FISH probes.
  • FISH fluorescence in situ hybridization
  • the procedure has been designed to maximize tissue permeability for FISH when using DNA FISH probes.
  • FFPE Formalin fixed paraffin-embedded
  • Preparation involved the use of reagents Provided In Kit (Cat# 32-801210). Not provided in the kit are: absolute ethanol (EtoH), Hemo-De Clearing Agent (Scientific Safety Solvents Cat. #HD-150), purified water (distilled or deionized), Coplin jars (16 slides/8 slots capacity maximum), 37°C and 80°C water baths (one at 73°C for the probe assay).
  • step one (1) twice using fresh Hemo-De each time.
  • step one Dehydrate slides in 100% EtOH for 1 minute at ambient temperature. Repeat.
  • Fixation of the sample is performed to minimize tissue loss during sample denaturation. This procedure is highly recommended when processing samples in a denaturation bath format, but is not necessary when processing slides using a Co- denaturation/Hybridization protocol.
  • the purpose of this procedure is to prepare human metaphase chromosome spreads and interphase nuclei on microscope slides for cytogenetic analysis and to prepare chromosome preparations for FISH/ISH hybridization procedures.
  • Fixative Methanol: glacial acetic acid, 3: 1. Prepare before each use.
  • Slides Label each Superfrost Plus slide accordingly on its frosted surface and place the slides in a rectangular staining dish with glass cover. Fill the dish with distilled water and soak at 4°C prior to use to chill slides. This can be done days in advance, and slides can be stored at 4°C.
  • lymphocyte cell pellet in fresh fixative in a 15mL conical tube. If the pellet was stored after its harvest, centrifuge it at 200 x g for 5 minutes. Aspirate the supernatant, and add sufficient fixative to make the cell suspension appear slightly cloudy. Cell concentration varies between cases and should be empirically determined.
  • Ethanol Series Prepare v/v dilutions of 100% ethanol with purified H2O. Between uses, store tightly covered at ambient temperature. Discard stock solutions after 6 months. Prepare 70%>, 85% and 100% ethanol using distilled water in plastic Coplin jars.
  • 2 X SSC Mix thoroughly 100 mL 20 X SSC (pH 5.3) with 850 mL purified H20. Measure pH and adjust to pH 7.0 ⁇ 0.2 with NaOH. Add purified H20 to bring final volume to 1 liter. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated. Prepare 2 X SSC in plastic coplin jar and preheat to 37oC using a water bath.
  • the resulting metaphase cells should have minimal overlaps and no visible cytoplasm, with chromosomes appearing as medium gray to dark gray under phase contrast microscopy.
  • chromosomes become distorted and puffy in appearance. If slides are aged extensively, hybridization efficiency decreases because the chromosomes are too hard.
  • the purpose of this protocol is to culture and harvest human lymphocytes to determine structural and numerical chromosomal abnormalities and to prepare chromosome preparations for FISH/ISH hybridization procedures.
  • PB-MAX Karyotyping Medium (IX): Thaw PB-MAX Karyotyping medium at 4°C to 8°C. Warm the medium to room temperature and gently swirl to mix prior to use. PB-MAX Karyotyping medium can be thawed and aseptically transferred into smaller aliquots for convenience. These aliquots can be frozen and thawed at time of use, however multiple freeze-thaw cycles should be avoided. Avoid prolonged exposure to light when using this culture medium product.
  • Fixative Methanol: glacial acetic acid, 3: 1. Prepare before each use.
  • PB-MAX Karyotyping Medium is composed of a liquid RPMI-1640 medium that is completely supplemented with standard concentrations of L-glutamine, gentamicin sulfate, fetal bovine serum and phytohemagglutinin. This formulation is based on Peripheral Blood Media referenced in ACT Laboratory manual (1991) for use in PHA stimulated Peripheral Blood Culture.
  • Hypotonic treatment causes a swelling of the cells; the optimal time of treatment varies for different cell types and must be determined empirically.
  • Labeled CEP Chromosome Enumeration Probes
  • DNA probes can be used to identify human chromosomes in metaphase spreads and interphase nuclei with fluorescence in situ hybridization (FISH) for example to identify aneuploidies in normal and tumor cells, to serve as reference probe in cytogenetic studies and to identify the human chromosomes in hybrid cell lines.
  • FISH fluorescence in situ hybridization
  • CEP 17 (D17Z1) SpectrumGreen Probe Abbott 06J37-027
  • 2X SSC solution Mix thoroughly 100 mL 20X SSC (pH 5.3) with 850 mL purified H2O. Measure pH and adjust to pH 7.0 ⁇ 0.2 with NaOH. Add purified H2O to bring final volume to 1 liter. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated. Prepare 2 X SSC in plastic coplin jar and preheat to 37°C using a water bath.
  • Denaturation Solution (70% Formamide/2X SSC): Mix thoroughly 49 mL ultrapure formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2O in a glass coplin jar. Measure pH using pH indicator strips to verify pH is 7.0-8.0. Between uses, store covered at 2-8 °C. Discard after 7days. Prepare in glass coplin jar and heat to 73+/-l°C.
  • Ethanol Solutions (70%, 85%, 100%): Prepare v/v dilutions of 100% ethanol with purified H2O. Between uses, store tightly covered at ambient temperature. Discard stock solutions after 6 months. Prepare 70%>, 85% and 100% ethanol using distilled water in plastic coplin jars.
  • Probe Signal Intensity The signal should be bright, distinct, and easily evaluable. Signals should be in either bright, compact, oval shapes or stringy, diffuse, oval shapes.
  • Cross-hybridization/Target Specificity The probe should hybridize and illuminate only the target (centromere of chromosome). Metaphase spreads should be evaluated to verify locus specificity and to identify any cross-hybridization to non-target sequences. At least 98% of cells should show one or more signals for acceptable hybridization. Signal Enumeration-Selection of optimum viewing area and evaluable nuclei
  • [0312] Use a 25X objective to scan the hybridized area and examine the specimen distribution. Select an area where the specimen is distributed sparsely, few interphase nuclei are overlapping, and several interphase nuclei can be scanned within a viewing field. Avoid areas where the distribution of cells is dense, cells are overlapped, or the nuclear border of individual nuclei is unidentifiable. Avoid areas that contain clumps of cells. Enumerate only those cells with discrete signals.
  • the purpose of this protocol is to perform FISH using LSI (Locus Specific Identifier) probes on cytogenetic specimens.
  • Labeled LSI DNA probes can be used to identify human chromosomes in metaphase spreads and interphase nuclei, and genetic aberrations with fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • the LSI BCR/ABL probe set is designed to detect fusion of the ABL gene locus on 9q34 and BCR gene locus on 22ql 1.2 (Translocation (9;22)(q34;ql 1)).
  • 2X SSC solution Mix thoroughly 100 mL 20X SSC (pH 5.3) with 850 mL purified H2O. Measure pH and adjust to pH 7.0 ⁇ 0.2 with NaOH. Add purified H2O to bring final volume to 1 liter. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated. Prepare 2 X SSC in plastic coplin jar and preheat to 37°C using a water bath.
  • Denaturation Solution (70% Formamide/2X SSC :Mix thoroughly 49 mL ultrapure formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2O in a glass coplin jar. Measure pH using pH indicator strips to verify pH is 7.0-8.0. Between uses, store covered at 2-8 °C. Discard after 7days. Prepare in glass coplinjar and heat to 73+/-l°C.
  • 0.4X SSC/0.3% NP-40 Wash Solution Mix thoroughly 20 mL 20X SSC (pH 5.3) with 950 mL purified H2O. Add 3 mL of NP-40. Mix thoroughly until NP-40 is completely dissolved. Measure pH and adjust pH to 7.0-7.5 with NaOH. Add purified H20 to bring final volume of the solution to 1 liter. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or
  • Ethanol Solutions (70%, 85%, 100%): Prepare v/v dilutions of 100% ethanol with purified H2O. Between uses, store tightly covered at ambient temperature. Discard stock solutions after 6 months. Prepare 70%>, 85% and 100% ethanol using distilled water in plastic coplin jars.
  • the Triple bandpass filter DAPI/FITC/Texas Red is optimal for viewing all three fluorophores simultaneously. Evaluate slide adequacy using the following criteria:
  • Probe Signal Intensity The signal should be bright, distinct, and easily evaluable. Signals should be in either bright, compact, oval shapes or stringy, diffuse, oval shapes.
  • Cross-hybridization/Target Specificity The probe should hybridize and illuminate only the target. Metaphase spreads should be evaluated to verify locus specificity and to identify any cross-hybridization to non-target sequences. Selection of optimum viewing area and evaluable nuclei
  • [0355] Use a 25X objective to scan the hybridized area and examine the specimen distribution. Select an area where the specimen is distributed sparsely, few interphase nuclei are overlapping, and several interphase nuclei can be scanned within a viewing field. Avoid areas where the distribution of cells is dense, cells are overlapped, or the nuclear border of individual nuclei is unidentifiable. Avoid areas that contain clumps of cells. Analyze only those cells with discrete signals.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
  • any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

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Abstract

La présente invention concerne des biomarqueurs prédictifs et des méthodes d'utilisation pour la détermination de la résistance et de la sensibilité à l'insuline, en plus d'une maladie cardiovasculaire et d'un risque associé à l'obésité. Des méthodes pour la stratification des patients le long du continuum de sensibilité à un risque cardiométabolique, comprenant la prédiction et l'évolution jusqu'au syndrome métabolique, sont également proposées.
PCT/US2014/051791 2013-08-20 2014-08-20 Biomarqueurs prédictifs pour syndrome métabolique Ceased WO2015026893A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11169145B2 (en) 2017-05-01 2021-11-09 Washington University Methods of detection and treatment for cardiovascular disease and foot wounds

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090304714A1 (en) * 2008-03-25 2009-12-10 The Regents Of The University Of Michigan IKKi Inhibitor Therapies and Screening Methods, and Related IKKi Diagnostics
US20120135927A1 (en) * 2009-05-21 2012-05-31 Joslin Diabetes Center, Inc. Bone morphogenetic proteins for the treatment of insulin resistance
US20120148588A1 (en) * 2009-06-08 2012-06-14 Acceleron Pharma, Inc. Methods for increasing thermogenic adipocytes
US20130109587A1 (en) * 2011-10-26 2013-05-02 Nuclea Biotechnologies, Inc. Usp2a peptides and antibodies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090304714A1 (en) * 2008-03-25 2009-12-10 The Regents Of The University Of Michigan IKKi Inhibitor Therapies and Screening Methods, and Related IKKi Diagnostics
US20120135927A1 (en) * 2009-05-21 2012-05-31 Joslin Diabetes Center, Inc. Bone morphogenetic proteins for the treatment of insulin resistance
US20120148588A1 (en) * 2009-06-08 2012-06-14 Acceleron Pharma, Inc. Methods for increasing thermogenic adipocytes
US20130109587A1 (en) * 2011-10-26 2013-05-02 Nuclea Biotechnologies, Inc. Usp2a peptides and antibodies

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11169145B2 (en) 2017-05-01 2021-11-09 Washington University Methods of detection and treatment for cardiovascular disease and foot wounds

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