US20110218116A1 - Biomarkers of osteoarthritis - Google Patents

Biomarkers of osteoarthritis Download PDF

Info

Publication number
US20110218116A1
US20110218116A1 US13/041,055 US201113041055A US2011218116A1 US 20110218116 A1 US20110218116 A1 US 20110218116A1 US 201113041055 A US201113041055 A US 201113041055A US 2011218116 A1 US2011218116 A1 US 2011218116A1
Authority
US
United States
Prior art keywords
subject
expression
biological sample
osteoarthritis
polypeptides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/041,055
Other languages
English (en)
Inventor
James L. Cook
Cristi R. Cook
Aaron M. Stoker
Keiichi Kuroki
Bridget Colleen Garner
Richard Evans
Brandon Lee Roller
Prakash Sidha Jayabalan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Missouri System
Original Assignee
University of Missouri System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=44531841&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20110218116(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by University of Missouri System filed Critical University of Missouri System
Priority to US13/041,055 priority Critical patent/US20110218116A1/en
Assigned to THE CURATORS OF THE UNIVERSITY OF MISSOURI reassignment THE CURATORS OF THE UNIVERSITY OF MISSOURI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COOK, CRISTI R., ROLLER, BRANDON LEE, COOK, JAMES L., EVANS, RICHARD, GARNER, BRIDGET COLLEEN, JAYABALAN, PRAKASH SIDHA, KUROKI, KEIICHI, STOKER, AARON M.
Publication of US20110218116A1 publication Critical patent/US20110218116A1/en
Priority to US14/175,254 priority patent/US9488646B2/en
Priority to US15/290,613 priority patent/US9733257B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5421—IL-8
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present disclosure relates to biomarkers of disease and more particularly to a plurality of biomarkers, related methods and kits for diagnosing, staging, and monitoring osteoarthritis.
  • Osteoarthritis is a debilitating disease that affects human and veterinary, particularly canine patients. Because OA is not typically diagnosed early enough to prevent the clinical progression of disease, development of early OA biomarkers has profound ramifications for diagnostic screening, disease staging, treatment planning and monitoring.
  • MCP1 monocyte chemoattractant protein 1
  • IL8 interleukin 8
  • KC keratinocyte derived chemoattractant
  • MMPs matrix metalloproteinases
  • the present disclosure provides a method for diagnosing, staging, or monitoring osteoarthritis in a subject comprising: measuring in a biological sample from the subject the level of expression of at least two polypeptides selected from the group consisting of: MCP1, IL8, KC, MMP2, MMP3, MMP9, IL6, MMP1, RANTES, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP, and fragments of any thereof, and any combination thereof, wherein the expression levels of the at least two polypeptides or fragments thereof in the biological sample provide a sample protein expression profile indicative of the presence or absence, degree, severity, type or stage of osteoarthritis in the subject.
  • the method may further comprise comparing the sample protein expression profile to a control protein expression profile, wherein a difference between the sample protein expression profile and the control protein expression profile is indicative of the presence or absence, degree, severity, type or stage of osteoarthritis in the subject.
  • the subject can be at risk of having or is suspected of having osteoarthritis.
  • the level of expression of the at least two polypeptides in the biological sample can be measured by many methods as detailed further herein below, including but not limited to detecting alterations in DNA due to a process selected from the group consisting of DNA amplification, DNA methylation/demethylation, and single nucleotide polymorphisms.
  • the present disclosure provides an OA biomarker expression profile comprising polypeptide expression level information for two or more polypeptides selected from the group consisting of: MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP and fragments of any thereof, and any combination thereof, obtained from a biological sample from a subject suspected of having osteoarthritis.
  • An OA expression profile may further comprise polypeptide expression level information for at least one biological sample obtained from at least one healthy subject.
  • Biological samples from the subject suspected of having osteoarthritis and the healthy subject or subjects may each comprise a sample of synovial fluid, a sample of whole blood, a sample of blood plasma, a sample of serum, a sample of urine, or a sample of saliva.
  • the biological samples are samples of synovial fluid.
  • Multiple biological samples of the same or different type may be obtained from each subject, OA expression level information obtained from each sample, and the results combined in a single OA expression profile.
  • the present disclosure provides a diagnostic reagent for osteoarthritis comprising two or more antibodies against any two or more OA biomarkers or fragments thereof selected from the group consisting of: MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP and fragments of any thereof.
  • the diagnostic reagent may be provided in a kit.
  • the present disclosure provides a kit for diagnosing osteoarthritis in a subject, the kit comprising: at least two OA biomarker detection reagents that each specifically bind to an OA polypeptide selected from the group consisting of MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP and fragments of any thereof, or at least two OA biomarker detection reagents that each specifically bind to at least part of a polynucleotide sequence coding for at least two of the OA polypeptides, wherein the specific binding of the reagent is indicative of the expression level of at least one OA polypeptide in a biological sample from a subject.
  • an OA polypeptide selected from the group consisting of MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apoli
  • the at least one reagent that specifically detects expression of at least one biomarker may comprise a nucleic acid probe complementary to at least part of a polynucleotide sequence coding for one of the polypeptides.
  • a nucleic acid probe can be a cDNA or an oligonucleotide.
  • the at least one OA biomarker detection reagent can be immobilized on a substrate surface.
  • the kit may comprise at least two biomarker detection reagents arranged on the substrate surface. In the kit, at least two biomarker reagents can be arranged on a substrate surface to comprise a microarray.
  • the present disclosure provides a method for identifying a candidate substance as a therapeutic agent for treating osteoarthritis, comprising: a) administering the candidate substance to a subject diagnosed with spontaneous osteoarthritis; b) measuring the expression level of two or more OA polypeptides selected from the group consisting of MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP in a biological sample from the subject; and c) selecting the candidate substance as a candidate therapeutic agent for treating osteoarthritis if the expression level of each of the two or more OA polypeptides in the biological sample is lower than or equal to the expression level for the selected two or more OA polypeptides in a biological sample from a control subject not administered the test substance.
  • the present disclosure provides a method for monitoring the effect of a treatment of osteoarthritis in a subject comprising: a) obtaining a first OA biomarker expression profile comprising measuring the expression level of two or more OA polypeptides selected from the group consisting of MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP in a first biological sample obtained from the subject before the osteoarthritis treatment is administered to the subject; b) obtaining a second OA biomarker expression profile comprising measuring the expression level of the two or more OA polypeptides selected in (a), in a second biological sample obtained from the subject after or while the osteoarthritis treatment is administered to the subject; and c) comparing the first OA biomarker expression profile with the second OA biomarker expression profile, wherein if the expression level of each of the two or more selected OA polypeptid
  • the biological sample or samples may comprise any one of synovial fluid, whole blood, blood plasma, serum, urine, and saliva.
  • the biological sample(s) comprise synovial fluid.
  • the level of expression of at least four polypeptides is measured.
  • the subject may be a mammal.
  • the subject is a human or a canine
  • the method may comprise measuring in a biological sample from the subject the level of expression of MCP1, IL8, KC, MMP2 and MMP3, or fragments thereof.
  • the subject is a human and the method may comprise measuring in a biological sample from the subject the level of expression of MCP1, IL6, IL8, KC and MMP1, or fragments thereof.
  • Such a method may further comprise measuring in the biological sample the level of expression of RANTES, or fragments thereof.
  • the expression level of each of the at least two polypeptides in the biological sample from the subject is measured using a method selected from the group consisting of: LUMINEX, ELISA, immunoassay, mass spectrometry, high performance liquid chromatography, two-dimensional electrophoresis, qPCR, RT-PCR, nucleic acid microarray, in situ hybridization, SAGE, Western blotting, protein microarray, and antibody microarray.
  • FIG. 1A is a bar graph showing levels (Mean ⁇ SE concentrations (pg/ml)), of Cartilage Intermediate Layer Protein (CILP), Decorin and COMP in dogs with subclinical OA and in dogs with clinical OA following removal of high abundance proteins.
  • CILP Cartilage Intermediate Layer Protein
  • FIG. 1B is a bar graph showing levels (Mean ⁇ SE concentrations (pg/ml)), of Apolipoprotein A1 and Apolipoprotein E in dogs with subclinical OA and in dogs with clinical OA following removal of high abundance proteins.
  • FIG. 2A is a bar graph showing levels (Mean ⁇ SE concentrations (pg/ml)), of IL8, KC, MCP1 and MMP3 in normal dogs and in dogs with spontaneous knee OA before and after surgery.
  • FIG. 2B is a bar graph showing levels (Mean ⁇ SE concentrations (pg/ml)), of MMP2 in normal dogs and in dogs with spontaneous knee OA before and after surgery.
  • FIG. 3 is a set of bar graphs summarizing (Mean ⁇ SE) MMP/cytokine log (pg/ml) levels in synovial fluid samples from normal patients and from patients with OA immediately preceding a total knee arthroplasty procedure.
  • antibody refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes, and encompasses polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as molecules engineered from immunoglobulin gene sequences.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • detecttable label refers to any moiety that generates a measurable signal via optical, electrical, or other physical indication of a change of state of a molecule or molecules coupled to the moiety.
  • Such physical indicators encompass spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, and chemical means, such as but not limited to fluorescence, chemifluorescence, chemiluminescence, and the like.
  • marker or “biomarker” as used interchangeably herein refer to any molecule used as a target for analyzing test samples obtained from subjects, and encompass proteins or polypeptides themselves as well as antibodies against same that may be present in a test sample. Proteins or polypeptides used as a marker include any variants and fragments thereof, and in particular, immunologically detectable fragments. For example, it is appreciated that variants of a marker polypeptide are encoded by the same gene, but can differ in their isoelectric point or molecular weight or both as a result of alternative processing such as alternative splicing and/or differences in post-translational modification (e.g., glycosylation, acylation, and/or phosphorylation).
  • alternative processing such as alternative splicing and/or differences in post-translational modification (e.g., glycosylation, acylation, and/or phosphorylation).
  • proteins or polypeptides used as a marker according to the present disclosure include fragments thereof.
  • certain markers can be synthesized in an inactive form that is subsequently converted to an active form by proteolysis. Proteins or fragments thereof can also occur as part of a complex. Proteins or polypeptides used as markers according to the present disclosure also include such complexes.
  • biomarker and “marker” also encompass nucleic acid molecules comprising a nucleotide sequence that codes for a marker protein, and also polynucleotides that can hybridize under stringent conditions with a part of such nucleic acid molecules.
  • OA biomarker and “OA marker” as used interchangeably herein, refer to a protein, polypeptide, antibodies against same and any fragment thereof, that may be present in a test sample from a subject, and has an expression level that has been found to be indicative of the presence of OA in the subject as described herein, and these terms also encompass any nucleic acid molecule comprising a nucleotide sequence that codes for an OA marker protein.
  • the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has or is currently undergoing any form of treatment.
  • the terms “subject” and “subjects” refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous monkey, chimpanzee, etc) and a human).
  • a mammal e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse
  • a non-human primate for example, a monkey, such as a cynomolgous monkey, chimpanzee, etc
  • the subject is a
  • test sample generally refers to a biological material being tested for and/or suspected of containing an analyte of interest.
  • the biological material may be derived from any biological source but preferably is a biological fluid likely to contain the analyte of interest.
  • biological materials include, but are not limited to, stool, whole blood, serum, plasma, red blood cells, platelets, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, soil, etc.
  • the test sample is a synovial fluid sample.
  • the test sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample.
  • pretreatment may include preparing plasma from blood, diluting viscous fluids and so forth. Methods of pretreatment may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc. If such methods of pretreatment are employed with respect to the test sample, such pretreatment methods are such that the analyte of interest remains in the test sample at a concentration proportional to that in an untreated test sample (e.g., namely, a test sample that is not subjected to any such pretreatment method(s)).
  • osteoarthritis refers to the disease also known as osteoarthrosis and degenerative joint disease, characterized by inflammation and damage to, or loss of cartilage in any joint or joints, and joint pain.
  • Clinical standards for diagnosing osteoarthritis in subjects including mammalian subjects such as canines and humans are well known and include for example swelling or enlargement of joints, joint tenderness or pain, decreased range of motion in joints, visible joint deformities such as bony growths, and crepitus. Symptoms can be identified by clinical observation and history, or imaging including MRI and X-ray.
  • Criteria for diagnosing the presence or absence of OA and severity or degree of OA include but are not limited to the ACR Criteria for knee OA (R. Altman et al., Development of criteria for the classification and reporting of osteoarthritis: Classification of osteoarthritis of the knee: Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. A RTHRITIS R HEUM. August 29(8):1039-1049(1986)), functional status criteria according to WOMAC (N. Bellamy et al., 1988, Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee.
  • expression refers to the conversion of the DNA sequence information into messenger RNA (mRNA) or protein. Expression may be monitored by measuring the levels of full-length mRNA, mRNA fragments, full-length protein, or protein fragments. Expression may also be inferred by assessing alterations in the DNA relative to a control state. Alterations in DNA that affect expression include amplification (increased copy number) of the DNA, changes in the methlyation status of the regulatory region of a gene, or single nucleotide polymorphisms in the regulatory region of a gene.
  • hybridization refers to the process of annealing or base-pairing via specific hydrogen bonds between two complementary single-stranded nucleic acids.
  • stringency of hybridization is determined by the conditions of temperature and ionic strength. Nucleic acid hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which the hybrid is 50% denatured under defined conditions. Equations have been derived to estimate the Tm of a given hybrid; the equations take into account the G+C content of the nucleic acid, the length of the hybridization probe, etc. (e.g., Sambrook et al, 1989, chapter 9).
  • hybridizations are generally carried out in solutions of high ionic strength (6 ⁇ SSC or 6 ⁇ SSPE) at a temperature that is about 20-25° C. below the Tm. If the sequences to be hybridized are not identical, then the hybridization temperature is reduced 1-1.5° C. for every 1% of mismatch.
  • the washing conditions are as stringent as possible (i.e., low ionic strength at a temperature about 12-20° C. below the calculated Tm).
  • highly stringent conditions typically involve hybridizing at 68° C. in 6 ⁇ SSC/5 ⁇ Denhardt's solution/1.0% SDS and washing in 0.2 ⁇ SSC/0.1% SDS at 65° C.
  • the optimal hybridization conditions generally differ between hybridizations performed in solution and hybridizations using immobilized nucleic acids. One skilled in the art will appreciate which parameters to manipulate to optimize hybridization.
  • nucleic acid molecule and “polynucleotide” as used interchangeably herein, refer to sequences of linked nucleotides.
  • the nucleotides may be deoxyribonucleotides or ribonucleotides, they may be standard or non-standard nucleotides; they may be modified or derivatized nucleotides; they may be synthetic analogs.
  • the nucleotides may be linked by phosphodiester bonds or non-hydrolyzable bonds.
  • the nucleic acid may comprise a few nucleotides (i.e., oligonucleotide), or it may comprise many nucleotides (i.e., polynucleotide).
  • the nucleic acid may be single-stranded or double-stranded.
  • protein refers to molecules composed of multiple amino acids having sequences of a variety of lengths acid including the full-length native protein or a shorter fragment of the full-length protein. These may be in neutral forms or as salts, either unmodified or modified by processes including glycosylation, side chain oxidation, and phosphorylation, or by the addition of other moieties attached to amino acid side chains, including but not limited to glycosyl units, lipids, and inorganic ions such as phosphates. Modifications may also include chemical conversion of amino acid side chains, such as oxidation of sulfhydryl groups.
  • Protein and its equivalents as used herein, encompasses protein isoforms encoded by the same gene that may differ in pI, MW, or both. Such isoforms may have different amino acid sequences resulting, for example, from differential processing such as alternative splicing, or post-translational modification(s).
  • protein fragment refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a second polypeptide.
  • the fragment of a marker protein may or may not possess a functional activity of the full-length native protein.
  • the methods described herein, and diagnostic reagents, kits and related inventions disclosed herein are based in part on the surprising discovery of a plurality of molecular markers, the expression levels of which consistently differentiate between healthy subjects and subjects with OA.
  • the same plurality of markers is able to distinguish between pre- and post-surgical OA subjects, thus also indicating that OA treatment efficacy can be evaluated with these markers.
  • the molecular markers are genes whose altered expression in subject, as measured from a readily obtained biological sample from the subject, is indicative of the presence of OA in the subject.
  • methods of using the molecular markers to identify a candidate substance as a therapeutic substance for OA treatment methods for determining OA treatment efficacy in a subject, and OA diagnostic reagents and kits.
  • an “OA biomarker” is indicative of OA when the expression level or quantity or structure of the biomarker is found significantly more often in subjects with OA present, or having OA of the same degree, severity, type or stage, than in subjects without OA, or lacing OA of the same degree, severity, type or stage.
  • Significance of an expression level, quantity or structure of the OA marker, as compared to a control, is determined using routine statistical methods by applying accepted confidence levels, e.g. at a minimum of 95%. It will be understood, for example, that cut-off or threshold expression levels for each OA biomarker may set according to many factors including the degree of correlation of expression level with clinical or subclinical OA indicators.
  • an expression level of a biomarker that is indicative of OA can be, for example, that found in at least 60% of patients who have the disease and is found in less than 10% of subjects who do not have the disease. More preferably, an expression level is indicative of OA if found in at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or more in subjects who have the disease and is found in less than 20%, less than 10%, less than 8%, less than 5%, less than 2.5%, or less than 1% of subjects who do not have the disease.
  • An “OA expression profile” is any physical representation of the expression levels of a set of two or more selected OA markers, as determined from one or more biological samples from one or more subjects known to have OA, known to have OA of a particular type (subtype I or subtype II), known to have OA of a particular stage (early or late), or known to be free of OA.
  • a profile for a particular subject or group of subjects may include expression level information from multiple types of biological samples that have been analyzed separately for OA marker expression levels.
  • an OA expression profile for a subject may include OA marker expression level information from a urine sample and a blood sample from the subject, and the results combined in a single profile representing the OA marker expression levels from both samples.
  • a single expression profile may include expression level information from any two or more biological samples selected from synovial fluid, whole blood, blood plasma, serum, urine, and saliva, and a complete profile may include expression level information from any three or four biological samples selected from synovial fluid, urine, saliva, and whole blood, blood plasma or serum.
  • the profile may be represented in visual graphical form, for example on paper or on a computer display; in a three dimensional form such as an array; and/or stored in a computer-readable medium.
  • An expression profile may correspond to a particular status of OA (e.g., presence or absence of OA disease, severity (clinical or subclinical)), type (subtype I or subtype II OA), degree (degree of cartilage damage) or stage (early OA or late OA), and thus provides a template for comparison to a patient sample.
  • Control profiles can be obtained by analyzing a biological sample from at least one normal/healthy subject, or multiple samples obtained from a group of normal/healthy subjects, or from one or more subjects identified as having comparable OA disease in terms of severity, type or stage. Similarly, comparable profiles can be obtained for age-, sex- and body mass index-matched subjects.
  • normal and “healthy” are used herein interchangeably to refer to a subject or subjects who do not display and have no history of OA symptoms such as joint pain, inflammation, or decrease in function, and have not been diagnosed with OA.
  • a “normal” or “healthy” sample refers to a sample or samples obtained from a normal/healthy subject.
  • a “subject suspected of having OA” is a subject that exhibits one or more symptoms indicative of the presence of OA in the subject, such as but not limited to joint pain, joint swelling, and crepitus, or a subject that may be at risk of, or simply being screened for the presence of OA.
  • Risk factors for developing OA are generally well known and include, for example, age, overweight or obesity, traumatic injury, breed, and/or family history.
  • An OA biomarker expression profile may for example comprise polypeptide expression level information for two or more polypeptides selected from the group consisting of: MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, ILL Apolipoprotein A1, Apolipoprotein E and fragments of any thereof, and any combination thereof, obtained from a biological sample from a subject suspected of having osteoarthritis. More specifically, by analyzing samples of synovial fluid obtained from healthy patients and from patients with early OA or late OA, it has been discovered that the polypeptides listed in Table 1 discriminate between normal subjects and subjects with OA.
  • the panel of markers (see Table 1) comprises MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP. These proteins are found to be up-regulated in synovial fluid samples of subjects with early osteoarthritis compared to synovial fluid samples of normal individuals.
  • altered expression levels of any one or more, preferably two or more of the OA markers described herein may be used to determine the presence or absence, degree, severity, type or stage of OA in one or more subjects.
  • Altered expression of any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of the molecular markers may be used to determine the presence or absence, degree, severity, type or stage of OA in one or more subjects.
  • One skilled in the art will appreciate that, generally, the more markers examined, the more accurate the determination of the presence or absence, degree, severity, type or stage of OA in the one or more subjects.
  • Measuring the expression of any OA markers or a plurality of the OA markers may be accomplished by a variety of techniques that are well known in the art. Expression may be monitored directly by detecting products of the OA marker genes (i.e., mRNA or protein), or it may be assessed indirectly by detecting alterations in the DNA (e.g., amplification, methylation, etc.) that affect expression of the OA marker genes.
  • RNA, protein, or DNA may be isolated from cells of interest using techniques well known in the art and disclosed in standard molecular biology reference books, such as Ausubel et al., (2003) C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY, John Wiley & Sons, New York, N.Y.
  • RNA products of the OA marker genes may be accomplished by a variety of methods. Some methods are quantitative and allow estimation of the original levels of RNA between the OA sample and control sample, whereas other methods are merely qualitative. Additional information regarding the methods presented below may be found for example in Ausubel et al., (2003) C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY, John Wiley & Sons, New York, N.Y., or Sambrook et al. (1989) M OLECULAR C LONING: A L ABORATORY M ANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. A person skilled in the art will know which parameters may be manipulated to optimize detection of the mRNA of interest.
  • Quantitative real-time PCR may be used to measure the differential expression of any OA marker in an OA sample and control sample.
  • the RNA template is generally reverse transcribed into cDNA, which is then amplified via a PCR reaction.
  • the PCR amplification process is catalyzed by a thermostable DNA polymerase.
  • suitable thermostable DNA polymerases include Taq DNA polymerase, Pfu DNA polymerase, Tli (also known as Vent) DNA polymerase, Tfl DNA polymerase, and Tth DNA polymerase.
  • the PCR process may comprise three steps (i.e., denaturation, annealing, and extension) or two steps (i.e., denaturation and annealing/extension).
  • the temperature of the annealing or annealing/extension step can and will vary, depending upon the amplification primers. That is, their nucleotide sequences, melting temperatures, and/or concentrations.
  • the temperature of the annealing or annealing/extending step may range from about 50° C. to about 75° C.
  • the amount of PCR product is followed cycle-by-cycle in real time, which allows for determination of the initial concentrations of mRNA.
  • the reaction may be performed in the presence of a dye that binds to double-stranded DNA, such as SYBR Green.
  • the reaction may also be performed with fluorescent reporter probes, such as TAQMAN® probes (Applied Biosystems, Foster City, Calif.) that fluoresce when the quencher is removed during the PCR extension cycle. Fluorescence values are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction. The cycle when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).
  • Ct threshold cycle
  • QRT-PCR is typically performed using an internal standard.
  • the ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. Suitable internal standards include, but are not limited to, mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and beta-actin.
  • Reverse-transcriptase PCR may also be used to measure the differential expression of an OA marker.
  • RNA template is reverse transcribed into cDNA, which is then amplified via a typical PCR reaction. After a set number of cycles the amplified DNA products are typically separated by gel electrophoresis. Comparison of the relative amount of PCR product amplified in the different cells will reveal whether the molecular marker is differentially expressed in an OA sample.
  • Differential expression of an OA marker may also be measured using a nucleic acid microarray.
  • single-stranded nucleic acids e.g., cDNAs, oligonucleotides, etc.
  • the solid support may be a material such as glass, silica-based, silicon-based, a synthetic polymer, a biological polymer, a copolymer, a metal, or a membrane.
  • the form or shape of the solid support may vary, depending on the application. Suitable examples include, but are not limited to, slides, strips, plates, wells, microparticles, fibers (such as optical fibers), gels, and combinations thereof.
  • the arrayed immobilized sequences are generally hybridized with specific DNA probes from the cells of interest.
  • Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest.
  • the probes are hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array.
  • Microarray analysis may be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.
  • RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked, and hybridized, under highly stringent conditions, to a labeled DNA probe. After washing to remove the non-specifically bound probe, the hybridized labeled species are detected using techniques well known in the art.
  • the probe may be labeled with a radioactive element, a chemical that fluoresce when exposed to ultraviolet light, a tag that is detected with an antibody, or an enzyme that catalyses the formation of a colored or a fluorescent product.
  • a comparison of the relative amounts of RNA detected in a control sample and a test sample will reveal whether the expression of the OA marker or OA markers is changed in the test sample.
  • Nuclease protection assays may also be used to monitor the differential expression of an OA marker in an OA sample and control sample.
  • an antisense probe hybridizes in solution to an RNA sample.
  • the antisense probe may be labeled with an isotope, a fluorophore, an enzyme, or another tag.
  • nucleases are added to degrade the single-stranded, unhybridized probe and RNA.
  • An acrylamide gel is used to separate the remaining protected double-stranded fragments, which are then detected using techniques well known in the art. Again, qualitative differences in expression may be detected.
  • Differential expression of an OA marker may also be measured using in situ hybridization.
  • This type of hybridization uses a labeled antisense probe to localize a particular mRNA in cells of a tissue section. The hybridization and washing steps are generally performed under highly stringent conditions.
  • the probe may be labeled with a fluorophore or a small tag (such as biotin or digoxigenin) that may be detected by another protein or antibody, such that the labeled hybrid may be visualized under a microscope.
  • the transcripts of an OA marker may be localized to the nucleus, the cytoplasm, or the plasma membrane of a cell.
  • Detection of the protein products of the OA markers may be accomplished by several different techniques, many of which are antibody-based. Additional information regarding the methods discussed below may be found in Ausubel et al., (2003) C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY, John Wiley & Sons, New York, N.Y., or Sambrook et al. (1989) M OLECULAR C LONING: A L ABORATORY M ANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. One skilled in the art will know which parameters may be manipulated to optimize detection of the protein of interest.
  • An enzyme-linked immunosorbent assay may be used to detect and quantify protein levels.
  • This method comprises preparing the antigen (i.e., protein of interest), coating the wells of a microtiter plate with the antigen, incubating with an antibody that recognizes the antigen, washing away the unbound antibody, and detecting the antibody-antigen complex.
  • the antibody is generally conjugated to an enzyme, such as horseradish peroxidase or alkaline phosphatase, which generate colorimetric, fluorescent, or chemiluminescent products.
  • An ELISA may also use two antibodies, one of which is specific to the protein of interest and the other of which recognizes the first antibody and is coupled to an enzyme for detection. Further, instead of coating the well with the antigen, the antibody may be coated on the well. In this case, a second antibody conjugated to a detectable compound is added following the addition of the antigen of interest to the coated well.
  • the Luminex platform (available from Luminex Corp., Austin, Tex.) can be used to detect and quantify protein levels using multiplexed assays based on a capture bead system in which microsphere beads are color-coded with dyes into up to one hundred distinct sets. Each color-coded bead set is coated with a specific binding reagent such as an antibody specific to a selected protein marker, allowing the capture and detection of specific protein analytes from a very small amount, e.g a drop of fluid, from a biological sample such as plasma, serum, lysates or synovial fluid.
  • a specific binding reagent such as an antibody specific to a selected protein marker
  • At least one or several bead sets may be incubated with the sample in order to capture the analytes.
  • a Luminex compact analyzer uses lasers to excite the internal dyes that identify each microsphere beads, and also any reporter dye captured during the assay. Multiple readings can be made on each bead set. Because of the special dye ratio incorporated each bead, each unique bead population can be analyzed separately after acquisition.
  • An exemplary multiplex immunoassay platform is also the xMAP platform available from Qiagen Inc.
  • Relative protein levels may also be measured by Western blotting.
  • Western blotting generally comprises preparing protein samples, using gel electrophoresis to separate the denatured proteins by mass, and probing the blot with antibodies specific to the protein of interest. Detection is usually accomplished using two antibodies, the second of which is conjugated to an enzyme for detection or another reporter molecule. Methods used to detect differences in protein levels include colorimetric detection, chemiluminescent detection, fluorescent detection, and radioactive detection.
  • Measurement of protein levels may also be performed using a protein microarray or an antibody microarray.
  • the proteins or antibodies are covalently attached to the surface of the microarray or biochip.
  • the protein of interest is detected by interaction with an antibody, and the antibody/antigen complexes are generally detected via fluorescent tags on the antibody.
  • Relative protein levels may also be assessed by immunohistochemistry, in which a protein is localized in cells of a tissue section by its interaction with a specific antibody.
  • the antigen/antibody complex may be visualized by a variety of methods. One or two antibodies may be used, as described above for ELISA.
  • the detection antibody may be tagged with a fluorophore, or it may be conjugated to an enzyme that catalyzes the production of a detectable product.
  • the labeled complex is typically visualized under a microscope.
  • Changes in the expression of any one or more OA markers may also be assessed by detecting alterations in the DNA encoding each OA marker gene.
  • the DNA may be amplified, which is a process whereby the number of copies of a region of DNA or a gene is increased. Usually, the amount of RNA product is also increased, in proportion to the number of additional copies of DNA.
  • Amplification of DNA may be detected by PCR techniques, which are well known in the art.
  • Amplification of DNA may also be detected by Southern blotting, in which genomic DNA is hybridized to labeled probes under highly stringent conditions, and the labeled hybrids may be detected as described above for Northern blotting.
  • Changes in the methylation status of DNA may also indicate changes in expression of any OA marker.
  • the regulatory region of a gene may be methylated, which entails the addition of a methyl group to the 5-carbon of cytosine in a CpG dinucleotide. Genes that are transcriptionally silent tend to have methylated or hypermethylated regulatory regions.
  • demethylation of an OA marker gene may lead to increased expression in tissue or cells, which is detectable from a biological sample obtained from a subject.
  • methylation of an OA marker gene may lead to decreased expression in issue or cells, which is detectable from a biological sample obtained from a subject.
  • Changes in the methylation status of an OA marker gene in a sample or samples from one or more subjects with OA, relative to a sample or samples from one or more control subjects, may be assessed using methylation-sensitive restriction enzymes to digest DNA followed by Southern detection or PCR amplification. Changes in the methylation status of an OA marker may also be detected using a bisulfite reaction based method. For this, sodium bisulfite is used to convert unmethylated cytosines to uracils, and then the methylated cytosines are detected by methylation specific PCR (MSP).
  • MSP methylation specific PCR
  • Single nucleotide polymorphisms (SNPs) in the regulatory region of an OA marker gene may also affect its level of expression.
  • an altered nucleotide may affect the binding of a transcription factor such that transcription is up-regulated or down-regulated.
  • the presence of a particular SNP may be detected by DNA sequencing.
  • a SNP may also be detected by selective hybridization to an oligonucleotide probe (i. e., it hybridizes to a sequence containing a particular SNP, but not to sequences without the SNP).
  • a particular SNP may also be detected using a PCR based technique or an oligonucleotide microarray based assay.
  • OA cell may be isolated from a subject known to have OA based on generally accepted clinical indicators, and expression of any OA marker may be examined in vitro.
  • the type of biopsy used to isolated cells can and will vary, depending upon the location and nature of the OA.
  • a sample of cells, tissue, or biological fluid such as synovial fluid, may be removed by needle aspiration biopsy.
  • a fine needle attached to a syringe is inserted through the skin and into the organ, tissue or joint capsule of interest. The needle may be guided to the region of interest using ultrasound or computed tomography (CT) imaging.
  • CT computed tomography
  • a vacuum is created with the syringe such that cells or fluid may be sucked through the needle and collected in the syringe.
  • a sample of cells or tissue may also be removed by incisional or core biopsy. For this, a cone, a cylinder, or a tiny bit of tissue is removed from the region of interest. This type of biopsy is generally guided by CT imaging, ultrasound, or an endoscope.
  • RNA, protein, or DNA may be extracted from any biological sample containing cells or tissue, to permit analysis of the expression level or levels of any one or more OA markers using methods described herein above.
  • Biopsied cells or tissue may also be embedded in plastic or paraffin, from which nucleic acids may be isolated.
  • the expression of an OA marker may also be performed in the biopsied cells or tissue in situ (e.g., in situ hybridization, immunohistochemistry).
  • OA marker may also be examined in vivo in a subject.
  • a particular mRNA or protein may be labeled with fluorescent dye, a bioluminescent marker, a fluorescent semiconductor nanocrystal, or a short-lived radioisotope, and then the subject may be imaged or scanned using a variety of techniques, depending upon the type of label.
  • a method for diagnosing, staging, or monitoring osteoarthritis can include, for example, measuring in a biological sample from the subject the level of expression of at least two polypeptides selected from the group consisting of: MCP1, IL8, KC, MMP2, MMP3, MMP9, IL6, MMP1, RANTES, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP, and fragments of any thereof, and any combination thereof, wherein the expression levels of the at least two polypeptides or fragments thereof in the biological sample provide a sample protein expression profile indicative of the presence or absence, degree, severity, type or stage of osteoarthritis in the subject.
  • the level of expression of the at least two polypeptides is measured using any of the above protein or nucleic acid quantification techniques, including but not limited to by detecting alterations in DNA due to a process selected from the group consisting of DNA amplification, DNA methylation/demethylation, and single nucleotide polymorphisms.
  • the method may further comprise comparing the sample protein expression profile to a control protein expression profile, wherein a difference between the sample protein expression profile and the control protein expression profile is indicative of the presence or absence, degree, severity, type or stage of osteoarthritis in the subject.
  • the OA biomarkers described herein are thus used in methods to detect the presence of OA in a subject, to study populations of subjects as to the occurrence of OA, and to evaluate OA treatment efficacy. Accordingly, the present disclosure provides methods for characterizing test samples obtained from a subject suspected of having OA, for diagnosing OA in a subject, for identifying the subtype of OA, and for assessing the advancement of OA in a subject. In such methods, the biomarkers' expression levels determined for a biological sample obtained from the subject are compared to the levels in one or more control samples.
  • the control samples may be obtained from a healthy subject (or a group of healthy subjects), from a subject (or group of subjects) with OA, from a subject (or group of subjects) with subtype I OA or subtype II OA, and/or from an subject (or group of subjects) with a specific stage of the disease (e.g., early OA or late OA).
  • the control expression levels of the biomarkers of interest are preferably determined from a significant number of individuals, and an average or mean is obtained.
  • the expression levels determined for the biological sample under investigation are compared to at least one expression profile for OA, as described above.
  • the OA biomarkers having expression levels that correlate with the presence or absence of OA, or OA degree, severity, type or stage, are attractive targets for the identification of new therapeutic agents (e.g., using screens to detect compounds or substances that inhibit or enhance the expression of these biomarkers). Accordingly, the present disclosure also provides methods for the identification of compounds or substances with the potential for effectively treating OA, or slowing OA progression.
  • the OA biomarkers can be readily applied in various screening methods, for example for identifying a candidate substance as a therapeutic agent for treating osteoarthritis.
  • Such a method may comprise, for example, a) administering the candidate substance to a subject diagnosed with spontaneous osteoarthritis; b) measuring the expression level of two or more OA polypeptides selected from the group consisting of MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP in a biological sample from the subject; and c) selecting the candidate substance as a candidate therapeutic agent for treating osteoarthritis if the expression level of each of the two or more OA polypeptides in the biological sample is lower than or equal to the expression level for the selected two or more OA polypeptides in a biological sample from a control subject not administered the test substance.
  • the methods may further include, for example, contacting a biological system that expresses at least one OA biomarker, with a candidate (test) substance for a time and under conditions sufficient for the candidate substance to change the expression of the at least one OA biomarker, and measuring a first OA biomarker expression level.
  • the method may further comprise maintaining the biological system for the same time and under the same conditions in the absence of the candidate substance, or after contacting the biological system with a control substance, then measuring a second OA biomarker expression level; and comparing the first and second OA biomarker expression levels, wherein a first OA biomarker expression level that is less than or greater than the second OA biomarker expression level is indicative that the candidate substance is a candidate therapeutic agent for treating OA.
  • Any candidate substance or a plurality of substances can be screened, including but not limited to synthetic and natural substances, and any combination of naturally occurring and synthetic substances. It should be understood that such screening can be performed using multiple OA biomarkers in parallel, which may be faciltated using any of many readily commercially available multiplex assays.
  • the method may further include generating an OA expression profile for the one or more OA biomarkers being evaluated, which can include for example expression information for each OA biomarker under the test and control conditions.
  • Such screening methods may be carried out using any type of biological system, such as but not limited to a cell or cells, a biological fluid, a biological tissue, or an animal. Methods can be carried out using any system capable of showing cartilage degeneration in response to the presence of induced or spontaneously occurring OA, including but not limited to an animal model, a whole body part such as a knee, hip or elbow, or a portion thereof. Assay and screening methods can be performed using cells grown in standard tissue culture. Preferably, such cells are mammalian, and more preferably of canine or human origin.
  • Cells may be primary cells, secondary cells, or immortalized cells and can be prepared by techniques well known in the art, including cells that are genetically engineered to contain or knock out a selected gene, or are available from well known commercial sources such as the American Type Culture Collection, Manassas, Va. Those of routine skill in the art can select a cell type or cell line according to generally recognized principles such as the objective of the assay, type and number of OA marker, drug being tested, and the like. Cells may be cultured using standard cell culture techniques, media and standards, such as growing and maintaining in a sterile environment at 37° C.
  • Any candidate substance identified by the screening methods can be further tested in assays that allow for the determination of the compound's properties in vivo.
  • Suitable animal models of osteoarthritis are well known in the art, including models of spontaneously occurring OA and OA induced by surgical instability or genetic modification. Animal models of naturally occurring OA occur in knee joints of guinea pigs, mice, and Syrian hamsters. Models of OA induced by surgical instability include medial meniscal tear in guinea pigs and rats, medial or lateral partial meniscectomy in rabbits, and medial partial or total meniscectomy or anterior cruciate transection in dogs. Transgenic mouse models are known. Other animal models of OA that can be used for validating a candidate substance identified as a potential OA therapeutic agents include many others described in detail in the literature known to those in the art.
  • the OA biomarkers can be applied in a method for monitoring the effect of a treatment of osteoarthritis in a subject.
  • a method may comprise, for example, a) obtaining a first OA biomarker expression profile comprising measuring the expression level of two or more OA polypeptides selected from the group consisting of MCP1, IL8, KC, MMP2, MMP3, IL6, MMP1, RANTES, MMP9, IL1B, Apolipoprotein A1, Apolipoprotein E, DCN, CILP and COMP in a first biological sample obtained from the subject before the osteoarthritis treatment is administered to the subject; b) obtaining a second OA biomarker expression profile comprising measuring the expression level of the two or more OA polypeptides selected in (a), in a second biological sample obtained from the subject after or while the osteoarthritis treatment is administered to the subject; and c) comparing the first OA biomarker expression profile with the second OA biomarker expression profile, wherein if the expression
  • OA treatment and “osteoarthritis treatment” as used interchangeably herein, refer to the application of, or administration of any therapeutic device or agent that reduces the expression levels in a subject of any combination of two or more of the OA biomarkers described herein, and/or that reduces or eliminates clinical symptoms of OA in a subject.
  • an effective treatment may be selected for treating a subject with OA. If for example the OA is determined to be responsive to a particular pharmaceutical agent, then a treatment comprising the agent may be given to the subject. If, however, the OA is determined to be non-responsive to the agent, then another treatment may be selected for the subject. Thus, determining the responsiveness of a subject before administering a treatment regime would spare subjects from potentially toxic or unhelpful treatments. Route of administration for any agent that is a candidate substance for treating OA will vary depending upon factors including the nature of the agent.
  • the route of administration may be intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, oral, perfusion, lavage, or direct injection.
  • the treatment regimen can and will vary, depending on the type of OA, its location, its stage, and the health and age of the subject.
  • Kits according to the present disclosure may comprise one or more reagents for measuring the expression of at least one OA marker, wherein changes in the expression of the one or more OA in a subject relative to a control subject are indicative of the presence or absence, stage, severity or subtype of OA.
  • OA markers comprise those listed in Table 1.
  • a diagnostic kit may include at least one reagent that is capable of specifically binding to at least one OA marker as described herein, to thereby detect the expression level of one or more of the OA markers.
  • Each kit may comprise one or more specific binding reagents, each binding reagent specific to a selected OA marker or fragment thereof.
  • the specific binding reagents may each comprise any molecule capable of such specific binding, such as an antibody that specifically binds to the protein marker, or fragment thereof, or a nucleic acid probe complementary to a polynucleotide sequence such as a cDNA or oligonucleotide.
  • specific binding is meant the reaction of the reagent with the polypeptide to produce a detectable product, while not reacting detectably with other polypeptides having unrelated sequences.
  • Specific binding reagents to be used in the measurement of the expression of the OA markers can and will vary, depending upon the type of technique to be used.
  • the kit may comprise oligonucleotide primers for QRT-PCR.
  • Nucleic acid probes contained may be included in a kit and are optionally provided together with a solid substrate, such as but not limited to beads, a chip, a plate, and a microarray.
  • Nucleic acid probes are optionally immobilized on the surface of such a substrate.
  • the kit may comprise fluorescent reporter probes.
  • the kit may also further comprise a reverse transcriptase, a Taq polymerase, and appropriate buffers and salts.
  • a kit may comprise antibodies that can be used for an immunoassay, e.g. for an ELISA.
  • the kit may further comprise a substrate for detection of enzyme-conjugated antibodies.
  • Antibodies that can be used in the methods and included in kits include monoclonal and polyclonal antibodies, immunologically active fragments (e.g., Fab or (Fab)2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, and chimeric antibodies.
  • Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras may be prepared and purified using methods well known in the art, or obtained from scientific or commercial sources.
  • Any binding agent can be directly or indirectly labeled with a detectable label.
  • the detectable label generates a signal that can be measured and is correlated, e.g. proportional to the amount of protein marker present in the sample being analyzed.
  • Detectable labels, methods for labeling molecules including polypeptides, antibodies and oligonucleotides are well-known in the art.
  • kits useful for analyzing biological samples, for example determining the presence or absence, degree, severity, type or stage of OA in a subject, may be provided in a kit.
  • the kit may further comprise one or more additional reagents such as, but not limited to, buffers such as extraction buffers, amplification buffers, hybridization buffers, immunodetection buffers, labeling buffers, or any equivalent reagent.
  • additional reagents such as, but not limited to, buffers such as extraction buffers, amplification buffers, hybridization buffers, immunodetection buffers, labeling buffers, or any equivalent reagent.
  • Reagents may be supplied in solid (e.g., lyophilized) or liquid form, and these may optionally be provided in individual packages using containers such as vials, packets, bottles and the like, for each individual reagent.
  • Each component can for example be provided in an amount appropriate for direct use or may be provided in a reduced or concentrated form that can be reconstituted.
  • Diagnostic and treatment monitoring kits can further comprise materials and tools useful for carrying out diagnostic and monitoring methods according to the present disclosure.
  • the kits can be used for example in diagnostic laboratories, clinical or research settings.
  • the kit may further comprise instructions for use, including for example any procedural protocols and instructions for using the various reagents in the kit for performing different steps of the process.
  • Instructions for using the kit according to one or more methods of the invention may comprise instructions for processing the biological sample obtained from the subject and/or for performing the test, and instructions for analyzing or interpreting the results. Instructions may be provided in printed form or stored on any computer readable medium including but not limited to DVDs, CDs, hard disk drives, magnetic tape and servers capable of communicating over computer networks.
  • a kit may further comprise one or more control samples.
  • a kit may comprise at least one expression profile for OA, OA subtype, and/or OA progression as described herein for use as comparison template.
  • the expression profile is provided as digital information stored on a computer-readable medium.
  • Two 4mm explants were created from the tissue of each animal and incubated in 500 ⁇ l of DMEM with supplemental nutrients for 7 days.
  • Culture media from each individual was analyzed using a canine cytokine and chemokine immunoassay for MCP1, IL-8 and KC (Millipore) based on the xMAP platform.
  • a second aliquot was analyzed using a multiplex human MMP immunoassay for MMPs 2, 3 and 13 (R&D Systems) that has been shown to cross react with canine samples.
  • Clinically relevant subgroups were then created based on OA-status and the media from each subgroup was pooled for proteomics analysis.
  • Each media pool was acetone precipitated and quantified to ensure equivalent protein loading for one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) with reducing conditions. Gel separation of the normal group could not be pursued due to insufficient volume and very low protein concentration of the normal culture media.
  • Subclinical OA versus Severe Clinical OA ( FIG. 1 ): The bar graphs show quantitative values obtained by Mass-Spec for select proteins from subclinical OA and clinical OA (high) groups following the removal of high abundance blood proteins. Alterations of 155 proteins were identified in media between subclinical OA and severe clinical OA groups once high abundance proteins were masked. Cartilage Intermediate Layer Protein (CILP), Decorin and COMP were all lower in the severe clinical OA group [4.2 ⁇ , 4.0 ⁇ and 1.4 ⁇ respectively] ( FIG. 1A ). Apolipoproteins A1 and E were 9.1 ⁇ and 2.6 ⁇ higher in the severe clinical OA group, respectively ( FIG. 1B ).
  • CILP Cartilage Intermediate Layer Protein
  • FIG. 1A Apolipoproteins A1 and E were 9.1 ⁇ and 2.6 ⁇ higher in the severe clinical OA group, respectively.
  • the non-operated, contralateral hind limb served as an internal control for each dog.
  • Synovial fluid was collected immediately prior to surgery on the operated limb, and 12 weeks post-operatively from both the operated and contralateral control limbs. These were analyzed using a multiplex canine cytokine and chemokine immunoassay (Millipore) for IL-2, IL-4, IL-7, IL-8, IL-15, IL-18, IP-10, INF- ⁇ , TNF- ⁇ , MCP1, KC, and GM-CSF based on the xMAP platform. Results were statistically evaluated with the unpaired t-test or the Mann-Whitney Rank Sum test with significance set at p ⁇ 0.05. Imaging studies (including ultrasound and magnetic resonance imaging), arthroscopy and histopathologic data were also collected to fully characterize the pathology of all joint tissues.
  • AUC areas under the curve
  • MCP1/CCL2 monocyte chemoattractant protein 1
  • IL-8/CXCL8 interleukin-8
  • KC/CXCL1 keratinocyte-derived chemoattractant
  • the control group was comprised of nine medium and large breed adult dogs ranging from 2-5 years old (mean 2.9 years). These dogs had no clinical history of joint trauma, were not lame and were deemed to be free of clinical OA by a board certified veterinary orthopaedic surgeon. Radiographic evaluation of the shoulders, knees and hips verified the absence of OA. Blood and synovial fluid were collected in a similar manner to the OA dogs at a time convenient to the clients.
  • synovial fluid samples were analyzed in duplicate using a multiplex canine cytokine and chemokine immunoassay (Millipore Corp. St. Louis, Mo., USA) based on the xMAP platform (Qiagen Inc.) for IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IP-10, INF- ⁇ , TNF- ⁇ , MCP1, KC, and GM-CSF per manufacturers' instructions.
  • a multiplex canine cytokine and chemokine immunoassay (Millipore Corp. St. Louis, Mo., USA) based on the xMAP platform (Qiagen Inc.) for IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IP-10, INF- ⁇ , TNF- ⁇ , MCP1, KC, and GM-CSF per manufacturers' instructions.
  • MMP matrix metalloproteinase
  • Results Results obtained following the methods described above are summarized in FIGS. 2A and 2B and Table 3 (below).
  • MMP3 was highest in the Pre-Op OA dogs and lowest in the Post-Op OA dogs, but these changes did not reach statistical significance. ( FIGS. 2A and 2B ).
  • the remaining serum and SF were separated with 1D-PAGE and analyzed by liquid chromatography mass spectrometry (LC-MS/MS). Matches were searched against the National Center for Biotechnology Information (NCBI) non-redundant database taxonomy-limited to dogs only. Significant differences between groups were represented by a p ⁇ 0.05 and a fold change>2.0.
  • NCBI National Center for Biotechnology Information
  • MMP2 and MMP3 were highest in the normal dogs and lowest in the Pre-Op OA dogs.
  • Significant differences were not detected between groups for IL8, KC, MCP1, IL18, IL2, IL7 or GMCSF, and the remaining analytes were below the limit of detection for the assay.
  • ROC curve analysis demonstrates for the first time that a biomarker panel measuring synovial fluid MCP1, IL8, KC, MMP2 and MMP3 has the ability to consistently differentiate normal healthy knees from knees in dogs with spontaneously occurring clinical OA.
  • MCP1 was significantly lower in the Post-Op OA dogs compared to their Pre-Op OA values, and IL8 and KC declined after treatment as well, indicating that this novel biomarker panel has great potential for clinical use in both diagnostic and treatment monitoring applications.
  • Equal volumes of hyaluronidase treated synovial fluid samples were analyzed using the Fluorokine MAP human MMP (MMP-1, -2,-9, and 013) and cytokine (Interleukin 1 ⁇ (IL-1 ⁇ ), IL-6, IL-8, Tumor necrosis factor- ⁇ (TNF- ⁇ ), Macrophage inflammatory protein 1 ⁇ (MIP-1 ⁇ ), MIP-1 ⁇ , Monocyte chemotactic protein 1 (MCP-1), RANTES) multiplex panels (R&D Systems).
  • MMP-1, -2,-9, and 013 cytokine
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • MIP-1 ⁇ Macrophage inflammatory protein 1 ⁇
  • MIP-1 ⁇ Monocyte chemotactic protein 1
  • RANTES Monocyte chemotactic protein 1
  • MMP-1, IL-6, IL-8, and RANTES were significantly higher in the synovial fluid of OA patients compared to normal patients, as shown in FIG. 3 .
  • MMP-9, MMP-13, IL-1 ⁇ , TNF- ⁇ , and MIP-1 ⁇ were below the detection limits of this assay for all patients.
  • novel OA biomarker panels for distinguishing and determining the severity of various forms of arthritis in dogs.
  • a starting hypothesis is that the novel OA biomarker panel can have high sensitivity and specificity (>0.9) and ROC data (>0.8) for determining the presence and severity of OA in clinical cohorts of canine patients.
  • the University of Missouri's VMTH admits well over 100 cases in each of these cohorts each year. Epidemiologic data is obtained and recorded in the medical record for each case.
  • Comfortable range of motion (CROM) in each stifle is determined by placing a goniometer on each limb such that one arm of the goniometer is aligned with the femur and one arm is aligned with the tibia with the rotation point centered at the joint line.
  • Each stifle is flexed and extended to the point allowable without definitive resistance or signs of pain from the dog.
  • the maximal flexion and extension angles is recorded. This procedure is repeated 3 times.
  • the CROM is determined by subtracting the mean flexion angle from the mean extension angle for each stifle.
  • Radiographic Assessment The dogs are sedated to obtain craniocaudal and mediolateral digital radiographic views of affected stifles.
  • the radiographs are scored by one investigator blinded to information regarding patient cohort and clinical signs, utilizing a subjective scoring system. (13, 31).
  • a subjective scoring system 13, 31.
  • nine regions of the stifle are evaluated and given a score from 0-3 based on severity of secondary radiographic changes associated with clinical OA in dogs. Therefore, each stifle receives a score ranging from 0-27.
  • Stifles scored from 0 to 4 are considered normal.
  • Stifles scored from 5 to 9 are considered to have mild OA.
  • Stifles scored from 10-18 are considered to have moderate OA, and stifles scored 19-27 are considered to have severe OA.
  • Arthroscopic Assessment Arthroscopic evaluation of affected stifles are performed using craniolateral and craniomedial portals. All articular surfaces of the patella, femur and tibia are examined and scored with respect to degree of articular cartilage damage (ICRS system). Meniscal, ligamentous, and synovial pathology are arthroscopically assessed and described in terms of nature, extent, and location. Dogs in the normal stifle group do not undergo arthroscopy.
  • ICRS system degree of articular cartilage damage
  • Biomarkers A small aliquot (25 ⁇ l) from each sample is thawed. For urine and synovial fluid, samples are centrifuged at 14,000 rpm for 10 minutes to pellet debris, and the supernatant removed. Synovial fluid samples are incubated with hyaluronidase (MP Biomedicals, LLC, Solon, Ohio) at 37° C. for 60 minutes to decrease viscosity.
  • hyaluronidase MP Biomedicals, LLC, Solon, Ohio
  • each of the samples is admixed with monoclonal antibody-charged, small (5.6 micron), polystyrene microspheres in a 96-well plate. Following an overnight incubation at 4° C., a polyclonal secondary antibody is added, as well as streptavidin-phycoerythrin. The median fluorescence intensity (MFI) is determined for each sample, and concentrations (pg/ml) obtained from a standard curve.
  • MFI median fluorescence intensity
  • Statistical Analyses Strength of correlations between each biomarker and combinations of biomarkers to detect presence and stage of disease is analyzed using a Pearson's Correlation. Sensitivity, specificity, and receiver operating characteristic curve (ROC) data is calculated for each biomarker and combinations of biomarkers to determine discriminatory potential between presence, type, and severity of disease for the various cohorts based on all outcome measures employed.
  • ROC receiver operating characteristic curve
  • Radiographic Assessment Knee radiographs are assessed using the Modified Kellgren-Lawrence scoring system (21) where the medial and lateral compartments is reviewed, scored for the severity of changes and then scores totaled.
  • Biomarkers A small aliquot (25 ⁇ l) from each sample is thawed. For urine and synovial fluid, samples are centrifuged at 14,000 rpm for 10 minutes to pellet debris, and the supernatant removed. Synovial fluid samples are incubated with hyaluronidase (MP Biomedicals, LLC, Solon, Ohio) at 37° C. for 60 minutes to decrease viscosity.
  • hyaluronidase MP Biomedicals, LLC, Solon, Ohio
  • each of the samples is admixed with monoclonal antibody-charged, small (5.6 micron), polystyrene microspheres in a 96-well plate. Following an overnight incubation at 4° C., a polyclonal secondary antibody is added, as well as streptavidin-phycoerythrin. The median fluorescence intensity (MFI) is determined for each sample, and concentrations (pg/ml) obtained from a standard curve.
  • MFI median fluorescence intensity
  • Statistical Analyses Strength of correlations between each biomarker and combinations of biomarkers to detect presence and stage of disease is analyzed using a Pearson's Correlation. Sensitivity, specificity, and receiver operating characteristic curve (ROC) data is calculated for each biomarker and combinations of biomarkers to determine discriminatory potential between presence, type, and severity of disease for the various cohorts based on all outcome measures employed.
  • ROC receiver operating characteristic curve

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
US13/041,055 2010-03-05 2011-03-04 Biomarkers of osteoarthritis Abandoned US20110218116A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US13/041,055 US20110218116A1 (en) 2010-03-05 2011-03-04 Biomarkers of osteoarthritis
US14/175,254 US9488646B2 (en) 2010-03-05 2014-02-07 Biomarkers of osteoarthritis
US15/290,613 US9733257B2 (en) 2010-03-05 2016-10-11 Biomarkers of osteoarthritis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33951110P 2010-03-05 2010-03-05
US13/041,055 US20110218116A1 (en) 2010-03-05 2011-03-04 Biomarkers of osteoarthritis

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/175,254 Continuation US9488646B2 (en) 2010-03-05 2014-02-07 Biomarkers of osteoarthritis

Publications (1)

Publication Number Publication Date
US20110218116A1 true US20110218116A1 (en) 2011-09-08

Family

ID=44531841

Family Applications (3)

Application Number Title Priority Date Filing Date
US13/041,055 Abandoned US20110218116A1 (en) 2010-03-05 2011-03-04 Biomarkers of osteoarthritis
US14/175,254 Active US9488646B2 (en) 2010-03-05 2014-02-07 Biomarkers of osteoarthritis
US15/290,613 Active US9733257B2 (en) 2010-03-05 2016-10-11 Biomarkers of osteoarthritis

Family Applications After (2)

Application Number Title Priority Date Filing Date
US14/175,254 Active US9488646B2 (en) 2010-03-05 2014-02-07 Biomarkers of osteoarthritis
US15/290,613 Active US9733257B2 (en) 2010-03-05 2016-10-11 Biomarkers of osteoarthritis

Country Status (6)

Country Link
US (3) US20110218116A1 (fr)
EP (2) EP2542701A4 (fr)
JP (2) JP5877799B2 (fr)
AU (1) AU2011222558C1 (fr)
CA (1) CA2792163A1 (fr)
WO (1) WO2011109738A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080026949A1 (en) * 2006-06-28 2008-01-31 Respiris, Inc. Apparatus, compositions, and methods for assessment of chronic obstructive pulmonary disease progression among rapid and slow decline conditions
WO2013033627A2 (fr) * 2011-09-01 2013-03-07 The Regents Of The University Of California Diagnostic et traitement de l'arthrite à l'aide de l'épigénétique
WO2013067358A3 (fr) * 2011-11-04 2015-06-25 Singulex, Inc. Détection de l'arthrose
WO2015108958A1 (fr) * 2014-01-14 2015-07-23 Anna Plaas Cible thérapeutique pour inflammation musculo-squelettique
US20160331339A1 (en) * 2015-05-15 2016-11-17 The Trustees Of Columbia University In The City Of New York Systems And Methods For Early Detection And Monitoring Of Osteoarthritis
RU2603484C2 (ru) * 2011-05-10 2016-11-27 Нестек С.А. Способы диагностирования дегенеративного поражения сустава
US9829493B2 (en) 2013-07-15 2017-11-28 Steven J. Svoboda Methods and apparatus for assessment of risk for joint injury
WO2021087116A1 (fr) * 2019-10-30 2021-05-06 Cd Diagnostics, Inc. Biomarqueurs de l'arthrose précoce
US11152103B1 (en) 2020-12-29 2021-10-19 Kpn Innovations, Llc. Systems and methods for generating an alimentary plan for managing musculoskeletal system disorders
CN113866323A (zh) * 2021-12-01 2021-12-31 太原理工大学 一种骨关节炎标志物检测试纸的制备方法

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2867679B1 (fr) * 2012-06-28 2018-07-18 Nordic Bioscience A/S Détermination du taux de renouvellement du cartilage pathologique
KR101848064B1 (ko) * 2015-02-27 2018-04-11 조선대학교산학협력단 퇴행성관절염에 대한 바이오마커 및 상기 바이오마커를 포함하는 퇴행성관절염 진단 또는 예후분석용 키트
CN105911291A (zh) * 2016-04-26 2016-08-31 上海中信亚特斯诊断试剂有限公司 一种尿液微量蛋白检测试剂盒及其检测方法
CN107918014A (zh) * 2017-11-22 2018-04-17 南宁科城汇信息科技有限公司 一种骨关节炎标志物确认
CN107727728A (zh) * 2017-11-22 2018-02-23 南宁科城汇信息科技有限公司 一种iTRAQ标记在骨关节炎模型中的应用
CN107907693A (zh) * 2017-11-22 2018-04-13 南宁科城汇信息科技有限公司 一种oa标志物的建立
KR102478584B1 (ko) * 2019-06-04 2022-12-19 이화여자대학교 산학협력단 퇴행성관절염 예방 또는 치료용 항-oscar 항체
WO2020246760A1 (fr) * 2019-06-04 2020-12-10 이화여자대학교 산학협력단 Anticorps anti-oscar pour la prévention ou le traitement de l'arthrose
WO2022031006A1 (fr) * 2020-08-05 2022-02-10 가톨릭대학교 산학협력단 Composition pour diagnostiquer l'arthrose, et procédé pour fournir des informations pour diagnostiquer l'arthrose
WO2023008978A1 (fr) * 2021-07-30 2023-02-02 아주대학교산학협력단 Composition ciblant le sncg pour le traitement ou le diagnostic d'une maladie dégénérative

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7662573B2 (en) * 2004-08-18 2010-02-16 The United States Of America As Represented By The Department Of Health And Human Services Methods for evaluating osteoarthritis risk

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003272465A1 (en) * 2002-09-12 2004-04-30 Chondrogene Inc. Identification of sequences particularly useful for the diagnosis and identification of therapeutic targets for osteoarthritis
JP2008547006A (ja) * 2005-06-17 2008-12-25 ザ ブライハム アンド ウイメンズ ホスピタル， インコーポレイテッド 変形性関節症のタンパク質プロフィール
TW200812582A (en) * 2006-04-06 2008-03-16 Astrazeneca Ab Medicaments
WO2009022179A2 (fr) * 2007-08-14 2009-02-19 Astrazeneca Ab Procédé
WO2009111450A2 (fr) * 2008-03-03 2009-09-11 Dyax Corp. Protéines de liaison à la métalloprotéinase 9
US20120190042A1 (en) * 2009-01-22 2012-07-26 Reuben Gobezie Protein biomarkers and therapeutic targets for osteoarthritis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7662573B2 (en) * 2004-08-18 2010-02-16 The United States Of America As Represented By The Department Of Health And Human Services Methods for evaluating osteoarthritis risk

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080026949A1 (en) * 2006-06-28 2008-01-31 Respiris, Inc. Apparatus, compositions, and methods for assessment of chronic obstructive pulmonary disease progression among rapid and slow decline conditions
US9885085B2 (en) * 2006-06-28 2018-02-06 University Of Utah Research Foundation Apparatus, compositions, and methods for assessment of chronic obstructive pulmonary disease progression among rapid and slow decline conditions
RU2603484C2 (ru) * 2011-05-10 2016-11-27 Нестек С.А. Способы диагностирования дегенеративного поражения сустава
WO2013033627A2 (fr) * 2011-09-01 2013-03-07 The Regents Of The University Of California Diagnostic et traitement de l'arthrite à l'aide de l'épigénétique
WO2013033627A3 (fr) * 2011-09-01 2013-05-02 The Regents Of The University Of California Diagnostic et traitement de l'arthrite à l'aide de l'épigénétique
WO2013067358A3 (fr) * 2011-11-04 2015-06-25 Singulex, Inc. Détection de l'arthrose
US9829493B2 (en) 2013-07-15 2017-11-28 Steven J. Svoboda Methods and apparatus for assessment of risk for joint injury
US10041955B2 (en) 2013-07-15 2018-08-07 Steven J. Svoboda Methods and apparatus for assessment of risk for joint injury
US10488421B2 (en) 2013-07-15 2019-11-26 Steven J. Svoboda Methods for reducing the risk of osteoarthritis of the knee
WO2015108958A1 (fr) * 2014-01-14 2015-07-23 Anna Plaas Cible thérapeutique pour inflammation musculo-squelettique
US11655506B2 (en) * 2014-01-14 2023-05-23 Rush University Medical Center Therapeutic target for musculoskeletal inflammation
US20160331339A1 (en) * 2015-05-15 2016-11-17 The Trustees Of Columbia University In The City Of New York Systems And Methods For Early Detection And Monitoring Of Osteoarthritis
WO2021087116A1 (fr) * 2019-10-30 2021-05-06 Cd Diagnostics, Inc. Biomarqueurs de l'arthrose précoce
US11152103B1 (en) 2020-12-29 2021-10-19 Kpn Innovations, Llc. Systems and methods for generating an alimentary plan for managing musculoskeletal system disorders
CN113866323A (zh) * 2021-12-01 2021-12-31 太原理工大学 一种骨关节炎标志物检测试纸的制备方法

Also Published As

Publication number Publication date
AU2011222558C1 (en) 2017-06-15
EP2542701A4 (fr) 2013-08-21
US20170030925A1 (en) 2017-02-02
JP2015079000A (ja) 2015-04-23
JP5980299B2 (ja) 2016-08-31
US20140155499A1 (en) 2014-06-05
JP2013521505A (ja) 2013-06-10
AU2011222558B2 (en) 2015-07-02
US9733257B2 (en) 2017-08-15
US9488646B2 (en) 2016-11-08
JP5877799B2 (ja) 2016-03-08
WO2011109738A1 (fr) 2011-09-09
AU2011222558A1 (en) 2012-10-18
EP3202919A1 (fr) 2017-08-09
CA2792163A1 (fr) 2011-09-09
EP2542701A1 (fr) 2013-01-09

Similar Documents

Publication Publication Date Title
US9733257B2 (en) Biomarkers of osteoarthritis
Hsueh et al. Synergistic roles of macrophages and neutrophils in osteoarthritis progression
Hsu et al. Lung tissues in patients with systemic sclerosis have gene expression patterns unique to pulmonary fibrosis and pulmonary hypertension
US7598052B2 (en) Expression profile of thyroid cancer
JP2015522246A (ja) 特発性肺線維症の予後予測、診断および処置の方法
Lambertz et al. Expression of aquaporin 5 and the AQP5 polymorphism A (-1364) C in association with peritumoral brain edema in meningioma patients
JP2016526888A (ja) 敗血症バイオマーカー及びそれらの使用
WO2010085606A1 (fr) Biomarqueurs protéiniques et cibles thérapeutiques pour l'arthrose
JP2012073274A (ja) 変形性関節症のタンパク質プロフィール
US8224579B2 (en) Method of diagnosing osteoarthritis
KR20120100938A (ko) 불임 및/또는 난자 품질을 평가하기 위한 방법 및 장치
Kerna et al. Association of ADAM12-S protein with radiographic features of knee osteoarthritis and bone and cartilage markers
Marshall et al. Blood-based biomarkers for detecting mild osteoarthritis in the human knee
KR20110014983A (ko) 심장 동종이식 만성 거부반응 진단방법
Kato et al. Large-scale gene expression profiles, differentially represented in osteoarthritic synovium of the knee joint using cDNA microarray technology
AU2015224468B2 (en) Biomarkers of osteoarthritis
Chu et al. The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data
US7811765B2 (en) Methods and compositions for determining predisposition to inflammation-mediated cardiovascular disease
CN116445606A (zh) 血清分子标志物comp在辅助诊断抑郁症中的应用
Aigner et al. cDNA arrays in degenerative arthritis research
JP2011177144A (ja) 化学物質の生体毒性の検出方法

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE CURATORS OF THE UNIVERSITY OF MISSOURI, MISSOU

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:STOKER, AARON M.;COOK, CRISTI R.;GARNER, BRIDGET COLLEEN;AND OTHERS;SIGNING DATES FROM 20110309 TO 20110323;REEL/FRAME:026024/0415

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION