WO2008150964A1 - Methods and kits for diagnosing and treating acute joint injury - Google Patents
Methods and kits for diagnosing and treating acute joint injury Download PDFInfo
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- WO2008150964A1 WO2008150964A1 PCT/US2008/065236 US2008065236W WO2008150964A1 WO 2008150964 A1 WO2008150964 A1 WO 2008150964A1 US 2008065236 W US2008065236 W US 2008065236W WO 2008150964 A1 WO2008150964 A1 WO 2008150964A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- 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
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- 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
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
Definitions
- the present invention provides methods, kits and reagents for selecting patients with acute non-autoimmune joint injury for treatment.
- the present invention provides a method of selecting a patient for treatment, wherein the patient is suffering from non-autoimmune acute joint pain, the method comprising: detecting the level of IL-6 in a biological sample from a joint, wherein the presence of IL-6 is indicative of a patient to be selected for treatment.
- the methods of the present invention further comprise detecting the level in the biological sample from the joint of at least one other cytokine selected from the group consisting of MCP-I, MIP- l ⁇ and IFN ⁇ .
- the one other cytokine is MCP-I .
- the one other cytokine is MIP-I ⁇ .
- the one other cytokine is IFN ⁇ .
- the biological sample from the joint is a fluid from the joint. In other embodiments, the biological sample from the joint is a lavage sample.
- IL-6 or IL-6 and at least one other cytokine are detected by way of an immunoassay. In some embodiments, IL-6 or IL-6 and at least one other cytokine are detected using a method of detection that comprises detecting the level of nucleic acid. In some embodiments, detecting the nucleic acid can be done using am amplification reaction. In some embodiments, the amplification reaction is a polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- the joint subjected to the methods of the present invention is a synovial joint.
- the joint subjected to the methods of the present invention is a knee.
- the methods of the present invention can be used on a shoulder.
- the methods of the present invention are performed on a wrist.
- the joint subjected to the methods of the present invention is an elbow.
- the joint subjected to the methods of the present invention is a hip.
- the methods of the present invention can be practiced on the small joints of a hand or a foot.
- the methods of the present invention further comprise treating the patient.
- the patient can be treated surgically.
- a treatment agent can be administered to the patient.
- the treatment agent administered to the patient can be an anti-inflammatory agent.
- the present invention provides a method of selecting a patient for treatment, wherein the patient is suffering from non-autoimmune acute pain of a joint, the method comprising: detecting the level of MCP-I in a biological sample from a joint, wherein the presence of MCP-I is indicative of a patient to be selected for treatment.
- the method of the present invention further comprises detecting the level of at least one other cytokine selected from the group consisting of IL-6, MIP-I ⁇ and IFN ⁇ .
- MCP-I and IL-6 are detected.
- MCP-I and MIP-I ⁇ are detected.
- IL-6 and IFN ⁇ are detected.
- prediction of success of surgery can also be determined based on the diagnostic presence of the cytokine biomarkers of the present invention. For example, a patients with elevated IL-6 or MCPl levels is more likely to have beneficial outcome from surgical treatment.
- MCP-I alone or MCP-I and at least one other cytokine from the group consisting of IL-6, MIP- l ⁇ and IFN ⁇ is detected in a fluid from a joint
- MCP-I alone or MCP-I and at least one other cytokine from the group consisting of IL-6, MIP- l ⁇ and IFN ⁇ is(are) detected in a lavage sample from a joint.
- MCP-I alone or MCP-I and at least one other cytokine from the group consisting of IL-6, MIP-I ⁇ and IFN ⁇ is(are) detected in an immunoassay.
- the nucleic acid coding for MCP-I or MCP-I and at least one other cytokine from the group consisting of IL-6, MIP- l ⁇ and IFN ⁇ is detected in the detection method, hi some embodiments, the nucleic acid is detected using an amplification reaction.
- the amplification reaction used as a method of detection is a polymerase chain reaction (PCR).
- the methods of the present invention can be practiced on a joint sample from any synovial joint.
- the joint is selected from the group consisting of knee, wrist, ankle, hip, elbow and shoulder.
- a blood sample e.g., serum or plasma
- a blood sample from the patient can be evaluated for the presence of IL-6, MCP-I, MIP-I ⁇ , or IFN ⁇ , either alone or in any combination.
- the presence of IL-6 and at least one cytokine selected from the group consisting of IL-6, MCP-I, INF ⁇ and MIP- l ⁇ can be detected.
- An increase in the level of cytokine in the blood sample in comparison to a control e.g., a patient that does not have an acute non-autoimmune joint injury, is indicative of a patient that is a candidate for treatment.
- a cytokine biomarker IL-6, MCP-I, MIP-I ⁇ , or IFN ⁇
- IL-6 can be detected in situ in the joint of a patient as described herein using in vivo imaging techniques.
- the methods of the present invention further comprise treating the patient.
- the patient can be treated surgically.
- the patient can be treated with an anti-inflammatory agent.
- the patient can be treated with an inhibitor of IL-6, MCP-I, INF ⁇ and/or MIP- l ⁇ such as an antibody.
- the present invention provides a kit comprising an antibody panel, wherein the antibody panel consists of an antibody to IL-6 and one or more antibodies to a cytokine selected from the group of MCP-I, INF ⁇ and MIP-I ⁇ .
- the antibody panel consists of an antibody to IL-6 and an antibody to MCPl.
- the antibody panel consists of an antibody to IL-6, an antibody to MCP-I and either an antibody to IFN ⁇ or an antibody to MIP-I ⁇ .
- the antibody panel consists of an antibody to IL-6, an antibody to MCP-I, an antibody to IFN ⁇ and an antibody to MIP-I ⁇ .
- the antibodies in the kit are on a continuous solid substrate.
- the antibodies in the kit are each provided on a separate solid surface.
- the kit further provides a device for extracting the biological sample from a joint.
- the extraction device provided in the kit is directly linked to a chamber comprising the antibody panel.
- Figure 1 depicts a comparison of the mean concentration levels of IL-6, MCP-I and MIP-I beta in operative knees, non-operative knees and normal control knees.
- Figure 2 depicts a comparison of mean IFN ⁇ levels in operative knees, non-operative knees and normal control knees.
- An operative knee is defined as a knee a physician deems requires surgical rectification.
- a non-operative knee is a contralateral side to an operated knee.
- acute pain or "acute inflammation of the joint” or “acute joint inflammation” or “acute joint injury (lesion)” or “acute chondral injury (lesion) "acute meniscal injury(lesion)” is used to refer to joint or chondral or cartilaginous or meniscal injury associated with pain for about 30 weeks or less.
- acute is used to differentiate such pain or inflammation from chronic disorders such as osteoarthritis or any other type of chronic disorder.
- a chronic disorder is a disorder associated with periods of pain longer than about 30 weeks or any damage to the joint that is associated with general, age-related deterioration of the joint.
- joint deterioration or "abnormal meniscal/chondral/joint anatomy” is used herein to refer to any abnormalities in the anatomy of the joint that can be visualized on an MRI and that are typically associated with the typical joint deterioration as a result of ageing.
- Patient refers to humans, other non-human primates and other mammals. Typically, a patient is a human. In some instances, the patient is simian, feline, canine, equine, porcine, bovine, ovine or caprine.
- joint refers to any diarthoidal (also called synovial) joints.
- joint thus refers to any joint containing bone, articular cartilage, a joint capsule, a synovial tissue lining, and lubricating synovial fluid inside the capsule.
- chondral refers to the cartilage components of a joint.
- meniscal refers to a component of the knee.
- the synovial joint is used here to refer to a shoulder or wrist or ankle or hip or elbow, or the small joints of the fingers or toes.
- biological sample refers to a cell or population of cells or a quantity of tissue, fluid or lavasate from the joint of a subject.
- a biological sample can comprise cells from cartilagenous tissue or can be free of cells.
- non-autoimmune as used herein when referring to acute joint inflammation is used to describe an inflammatory response or reaction that does not involve self-reactive antibodies. Rheumatoid arthritis is an example of autoimmune condition characterized by joint inflammation.
- antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
- the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the 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.
- the singular term "an antibody” as used herein is understood to encompass plural referents unless the context clearly indicates otherwise.
- the plurality of the antibodies can belong to the same antibody species, e.g., in the case of monoclonal antibodies, while in some cases different antibodies species are encompassed the by phrase "an antibody”, e.g., a polyclonal antibodies.
- An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
- Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
- the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
- Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases.
- pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2; a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
- the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
- the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g. , single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990)).
- agents When referring to treatment methods, antibodies that are chimeric, human, humanized or otherwise specific to the species to be treated are used.
- agent is used to describe a compound that has or may have a pharmacological activity. Agents include compounds that are known drugs, compounds for which pharmacological activity has been identified but that are undergoing further therapeutic evaluation, and compounds that are members of collections and libraries that are to be screened for a pharmacological activity.
- the term includes an organic or inorganic chemical such a peptide, protein, including antibodies, and small molecules and natural products.
- a "normal joint” or a “control joint” as used herein typically refers to a joint with an insignificant level of pain. The level of pain that might be present in a normal joint, typically does not impact the function or quality of the patient's life to the degree that the patient seeks medical care.
- a "normal” or “control joint” may be a joint subject to chronic pain lasting over 30 weeks such as, for example, an arthritic joint.
- cytokine biomarker or “cytokine marker” as used herein refers to the following cytokines and chemokines: IL-6, MIP-I ⁇ , MCP-I or IFN ⁇ .
- cytokine biomarker or “cytokine marker” refers to a polypeptide fragment of IL-6, MIP-I ⁇ , MCP-I or IFN ⁇ .
- Diagnostic levels of cytokine biomarkers as used herein refer to the presence of levels of IL-6, IFN ⁇ , MCP-I or MIP-I ⁇ that are statistically significantly elevated relative to a normal joint. In some instances, IL-6 alone is used as a diagnostic marker.
- MCP-I alone is used as a diagnostic biomarker.
- IFN ⁇ alone is used as a diagnostic marker.
- MIP-I ⁇ alone is used as a diagnostic marker.
- the presence or levels of IL-6 and any one or more of the other three cytokine biomarkers are used as diagnostic biomarkers.
- the presence or levels of MCP-I and any one or more of the other three cytokine biomarkers are used as diagnostic biomarkers.
- all four cytokine biomarkers are used in a diagnostic assay.
- immunoassay refers to an assay that uses an antibody or antibodies to specifically bind an antigen.
- the immunoassay is characterized by the use of specific binding properties of a particular antibody or antibodies to isolate, target, and/or quantify the antigen.
- Specific binding between a binding agent, e.g., an antibody and a protein, for instance, a biomarker cytokine, refers to the ability of a capture- or detection-agent to preferentially bind to a particular cytokine that is present in a mixture; e.g., a fluid from a joint. Specific binding also means a dissociation constant (K D ) that is less than about 10 ⁇ 6 M; preferably, less than about 10 "8 M; and, most preferably, less than about 10 ⁇ 9 M.
- K D dissociation constant
- the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
- level of cytokine biomarker in a biological sample as used herein typically refers to the amount of protein, protein fragment or peptide levels of the cytokine biomarker (for example, IFN ⁇ or IL-6 or MCPl or MIP- l ⁇ ) that is present in a biological sample
- a "level of cytokine biomarker” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
- the "level of cytokine biomarker mRNA" in a biological sample as used herein refers to the amount of mRNA encoding the cytokine biomarker (for example, IFN ⁇ or IL-6 or MCPl or MIP- l ⁇ ) that is present in a cell or a biological sample.
- a "level of cytokine biomarker mRNA” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
- isolated refers to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene.
- purified in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
- nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
- “Purify” or “purification” in other embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.
- a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, radiographic, immunochemical, chemical, or other physical means.
- useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
- the labels may be incorporated into nucleic acids, proteins and antibodies at any position. Any method known in the art for conjugating the antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.
- a "labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
- method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.
- nucleic acid probe or oligonucleotide is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
- a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
- the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not functionally interfere with hybridization.
- probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
- the probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
- Diagnosis or prognosis may be based at the genomic level, or at the level of RNA or protein expression.
- determining the functional effect is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a cytokine biomarker (e.g., IL-6, IFN ⁇ , MCPl or MIPl ⁇ ) protein sequence, e.g., functional, enzymatic, physical and chemical effects.
- a cytokine biomarker e.g., IL-6, IFN ⁇ , MCPl or MIPl ⁇
- Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the cytokine biomarker protein; measuring binding activity or binding assays, e.g. binding to antibodies or other ligands, and measuring cellular proliferation. Determination of the functional effect of a compound on joint injury can be performed using exemplary assays disclosed above.
- the functional effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels for cytokine biomarker sequences, measurement of RNA stability, identification of downstream or reporter gene expression, e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
- Inhibitors or “antagonist” or “modulators” of the cytokine biomarker polynucleotide and polypeptide sequences are used herein to refer to molecules of agents capable of inhibiting, inactivating or reducing the levels of the cytokine biomarkers.
- Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of cytokine biomarker proteins, e.g., antagonists.
- Inhibitors include polypeptide inhibitors, such as antibodies, soluble receptors and the like, as well as nucleic acid inhibitors such as siRNA or antisense RNA, genetically modified versions of the cytokine biomarker proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic cytokine biomarker antagonists, small chemical molecules and the like.
- Assays for detecting inhibitors include, e.g., expressing the cytokine biomarker protein in vitro, in cells, or cell membranes, applying putative antagonist compounds, and then determining the functional effects on cytokine activity, as described above.
- the present invention provides methods, reagents and kits for diagnosing and/or for the prognosis of non-autoimmune acute joint inflammation by detecting biomarkers in a sample obtained from an individual thought to be suffering from acute joint injury.
- the invention is based on the discovery that the presence and levels of certain biomarker cytokines, e.g., the presence and levels of IL-6 or MCPl can be used to diagnose acute inflammation of a joint.
- IL-6 and at least one other biomarker cytokine selected from the group consisting of MCP-I, MIP-I ⁇ and IFN ⁇ can be used to diagnose acute inflammation of a joint.
- MCP-I and at least one other biomarker cytokine selected from the group consisting of IL-6, MIP- l ⁇ and IFN ⁇ can be used to diagnose acute inflammation of the joint.
- the present invention provides methods to detect joint injury or methods for treating joint injury, e.g., by inhibiting the expression and/or activity of one or more of the biomarker cytokines.
- the methods of the invention typically involve detecting the presence of the biomarker cytokine(s) in a sample taken from a joint from a patient.
- the levels of the biomarker cytokines will be compared to a control value obtained from a normal, injury free joint of a subject or from a joint from a subject where the joint is not painful.
- the control sample can be taken from a non-injured joint of the same subject.
- the method of the present invention thus provides a criterion for the determination of acute joint injury in a patient to aid in selecting patients as candidates for treatment and/or surgery.
- the methods of the present invention provide additional criterions for selecting patients who have sustained joint injury regardless of whether such patients suffer from normal, age-related deterioration of the joint(s).
- patients are individuals who have been suffering from acute joint pain
- An individual suffering from acute pain is an individual who has been experiencing pain in a joint for 30 or 25 weeks or less.
- the patient may, for example, have been experiencing joint pain for 20 weeks or less; 15 weeks or less; 10 weeks or less; 8 weeks or less; 6 weeks or less; 4 weeks or less; 2 2eeks or less; or 1 week or less.
- the present invention allows for the differentiation of acute joint pain from chronic joint pain such as pain that is frequently associated with osteoarthritis or rheumatoid arthritis.
- the methods of the present invention can be practiced on patients regardless of their age and sex.
- patients are selected based on the pain they experience in the affected joint.
- patients are selected for cytokine level assessment based on a recent joint injury.
- a recent join injury indicates an injury that has been associated with pain for about thirty weeks or less.
- Candidate patients for the methods of the present invention include patients who experience pain or are suspected to have acute inflammation of a joint.
- the methods of the present invention are applicable to human or animal diarthrodial joints.
- a diarthrodial joint contains a bone, articular cartilage, a joint capsule, synovial tissue lining, and a lubricating synovial fluid inside the capsule.
- the methods of the present invention can be applied to a shoulder, a knee, a wrist, an ankle, an elbow, a hip or any of the finger or toe joints.
- cytokine biomarkers can be used to practice the methods of the present invention: IL-6, IFN ⁇ , MIP-I ⁇ and/or MCPl .
- the cytokine biomarkers used for the methods of the present invention are IL-6 alone or IL-6 and any combination of the following biomarkers: MCPl, MIP-I ⁇ , and IFN ⁇ .
- IL-6 and IFN ⁇ are used to practice the methods of the present invention.
- IL-6 and MIP-I ⁇ are used as the cytokine biomarkers to diagnose acute joint inflammation.
- IL-6 and MCPl are the cytokine biomarkers used to practice the methods of the present invention.
- IL- 6 and INF ⁇ are used with either of or both MCPl or MIP-I ⁇ to practice the methods of the present invention.
- the cytokine biomarkers used for the methods of the present invention are either MCP-I alone or MCP-I and any combination of the following biomarkers: IL-6, MIP-I ⁇ , and IFN ⁇ .
- MCP-I and IFN ⁇ are used to practice the methods of the present invention.
- MCP-I and MIP-I ⁇ are used as the cytokine biomarkers to diagnose acute joint inflammation.
- MCP-I and IL-6 are the cytokine biomarkers used to practice the methods of the present invention, hi some embodiments, MCP-I and INF ⁇ are used with either of or both IL-6 or MIP-I ⁇ to practice the methods of the present invention.
- MIP 1 ⁇ alone is used as a cytokine biomarker of the present invention.
- IFN ⁇ is used as a cytokine biomarker to practice the methods of the present invention.
- IL-6 also called IFN-beta-2
- IFN-beta-2 The biological significance of IL-6 (also called IFN-beta-2) lies in the fact that it is induced under conditions in which IFN -beta- 1 is not induced, as in metabolically stressed cells. Its induction by ILl and TNF ⁇ suggests that it may play a role as an autocrine mediator of some effects of these cytokines in inflammation and acute phase responses, as well as regulate cell proliferation.
- IL-6 is identical to B-cell differentiation factor (BSF2) and enhances proliferation in hybridoma/plasmacytoma cells (Sehgal et al, Science 235: 731-732 (1987)).
- BSF2 B-cell differentiation factor
- IL-6 The primary sequence of IL-6 deduced from the cDNA shows that it has 184 amino acids and is distinct from other interleukins (Hirano et al., Nature 324: 73-76 (1986). In addition to its antiviral activity, IL-6 elicits acute phase response in liver cells.
- An exemplary human amino acid IL-6 sequence can be found deposited under NCBI accession number NP_000591.
- Interferons were originally characterized as antiviral entities (Isaacs et al., Proc. Roy. Soc. London 147B: 268-273 (1957).
- Two major classes of acid-stable (type I) interferons have been recognized in humans: (1) leukocyte interferon, released by stimulated leukocytes, (2) and fibroblast interferon, produced by stimulated fibroblasts.
- the two classes of interferons differ not only immunologically but also in their target cell specificities, although both induce a virus-resistant state in human cells.
- Human interferons have been classified into 3 groups: alpha, beta, and gamma.
- alpha- and beta-IFNs are acid-stable, but they differ immunologically and in regard to some biologic and physiochemical properties.
- the gamma or immune IFNs which are produced by T lymphocytes in response to mitogens or to antigens to which they are sensitized, are acid-labile and serologically distinct from alpha- and beta-IFNs.
- An exemplary amino acid sequence for human IFN ⁇ has been deposited under accession number NP_000610.
- IFN gamma A variety of functions have been attributed to IFN gamma. For instance, it is well known that IFN gamma is a cytokine integral to the development of the ThI lineage of T cells. T-cell production of IFN ⁇ strongly suppresses osteoclastogenesis by interfering with the RANKL/RANK signaling pathway (Takayanagi et al., Nature 408: 600-605 (2000)). IFN ⁇ induces rapid degradation of the RANK adaptor protein, TRAF6, resulting in strong inhibition of the RANKL-induced activation of the transcription factor NFKB and JNK.
- TRAF6 tumor necrosis factor
- MIP l ⁇ is an 8-kD acidic protein that is upregulated upon stimulation in monocytes, T cells, and other lymphocytes. It belongs to the CC chemokine subfamily and directs the migration of specific subsets of leukocytes. MIP l ⁇ is encoded by 2 paralogous genes, ACT2 (CCL4) and LAGl (CCL4L), that are closely situated on the long arm of chromosome 17 (Modi et al., Immunogenetics 53: 543-549 (2001)). The proteins share a common length and are identical at 89 of 92 amino acids. The first 2 amino acid differences occur in the signal peptide, while the third is in the mature protein. Within the transcribed region, the genes differ at 25 of 662 nucleotides. The amino acid sequence deposited under accession number P13236 is an exemplary sequence for MlP-l ⁇ .
- CD8+ T cells normally synthesize MIP-1-beta (Kamin-Lewis et al., Nat. Acad. Sd. 98: 9283-9288 (2001)).
- This beta-chemokine is clearly synthesized by a major population of CD8+ T cells with a phenotype that is not consistent with cytotoxic T-lymphocyte effector function.
- Fibroblast-like synoviocytes obtained from rheumatoid arthritis and osteoarthritis patients exhibited expression of a functional IL2 receptor of intermediate affinity composed solely of IL2R ⁇ and IL2R ⁇ (Co ⁇ igall et al., J. Immun. 166: 4141-4147 (2001)).
- MCPl has been proposed to serve to recruit macrophages and perpetuate inflammation in the joints of patients with rheumatoid arthritis (Corrigall et al., J. Immun. 166: 4141-4147 (2001)).
- a representative sequence for human MCPl has been deposited under accession number AAQ75526.
- cytokine biomarkers IL-6, MIP-l ⁇ , MCPl and IFN ⁇ can be used to select a patient with acute joint injury for treatment.
- the cytokine biomarkers, IL-6 or IL-6 and one or more of the following cytokine biomarkers: MIP-I ⁇ , MCPl and IFN ⁇ can be used to diagnose acute inflammation of a joint, hi some instances, the cytokine biomarkers, MCPl or MCPl and one or more of the following cytokine biomarkers: MIP-I ⁇ , IL-6 and IFN ⁇ , can be used to diagnose acute inflammation of a joint, hi some embodiments, the presence or level of these cytokine biomarkers can be used to select a patient as candidate for treatment. In some other embodiments, the presence or levels of the cytokine markers can be used to determine the success during the course of or after treatment of an inflamed joint.
- Biological samples in which the cytokine markers can be detected include, for example, fluids from joints, hi some embodiments, biological samples include a tissue biopsy which may or may not have a liquid component, hi some instances, e.g., when fluid cannot be extracted from the joint, a lavage of the joint may be performed.
- a lavage involves using a physiologic compatible solution, e.g., saline.
- Methods for obtaining fluid from joint samples are well known to those of skill in the art.
- fluid or a lavage sample can be extracted or from a joint using a needle and a syringe.
- fluid can be extracted from joint using a flexible device with an absorbent material on the tip.
- the device can comprise a syringe with saline placed over a flexible sheath; the saline can then be injected and needle removed leaving the sheath behind.
- An absorbent material e.g., cotton
- the absorbent material can then be inserted through a sheath into the joint. The flexing of the joint at this point can allow the absorbent material to absorb the fluid.
- the absorbent material can be wet with a physiologic solution, e.g., saline, and upon extraction of the fluid from a joint, the tip of the absorbent material can be then directly applied to a testing strip or testing chamber.
- Immunoassays can be used to qualitatively or quantitatively analyze the cytokine biomarker levels, e.g., the levels of IL-6 or MIP-I ⁇ or MCP-I or IFN ⁇ in a biological sample.
- cytokine biomarker levels e.g., the levels of IL-6 or MIP-I ⁇ or MCP-I or IFN ⁇ in a biological sample.
- a general overview of the applicable technology can be found in a number of readily available manuals, e.g., Harlow & Lane, Cold Spring Harbor Laboratory Press, Using Antibodies: A Laboratory Manual (1999).
- assessment of cytokine expression and levels can be made based on the level of gene expression of the particular cytokines.
- RNA hybridization techniques for determining the presence and/or level of mRNA expression are well known to those of skill in the art and can be used to asses the presence or level of gene expression of the cytokine biomarkers of interest.
- the methods and kits of the present invention utilize selective binding partners of the cytokine biomakers IL-6, MIP-I ⁇ , MCPl and IFN ⁇ to identify the presence or determine the levels of the cytokine biomarkers in a biological sample.
- the selective binding partner to be used with the methods and kits of the present invention can be, for instance, an antibody.
- monoclonal antibodies to the particular cytokine biomarkers can be used.
- polyclonal antibodies to the particular cytokine biomarkers can be employed to practice the methods and in the kits of the present invention.
- cytokine biomarkers of the present invention are available and can be used with the methods and kits of the present invention.
- the RDI division of Fitzgerald Industries Inc (Fitzgerald Industries International, Inc., Concord, MA) offers a wide variety of antibodies: an exemplary goat anti -human IL6 antibody is offered under catalog number RDI-IL6abGPl ; an exemplary mouse anti-human MIP l ⁇ antibody is sold by USBiological (United States Biological, Swampscott, MA) is available for purchase under catalog number M 1202-35.
- a monoclonal anti human IFN ⁇ antibody produced in a mouse host is available from USBiological is available for sale under catalog number 17662-16Ml.
- Anti human MCPl antibody (polyclonal) from a chicken host is available from USBiological under catalog number M2749-97. These examples are meant to illustrate the availability of commercial antibodies to the cytokine biomarkers of the present invention and are in no way to be limiting. It is well know to those of skill in the art that the type, source and other aspects of an antibody to be used is a consideration to be made in light of the assay in which the antibody is used, hi some instances, antibodies that will recognize its antigen target (in this instance an epitope or multiple epitopes from IL6 or MIP l ⁇ or MCPl or IFN ⁇ ) on a Western blot might not applicable to an ELISA or ELISpot assay and vice versa.
- the antibodies to be used for the assays of the present invention can be produced using techniques for producing monoclonal or polyclonal antibodies that are well known in the art ⁇ see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975).
- Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice ⁇ see, e.g., Huse et al, Science 246:1275-1281 (1989); Ward et al, Nature 341 :544-546 (1989)).
- Such antibodies can be used for therapeutic and diagnostic applications, e.g., in the treatment and/or detection of any of the specific cytokine-associated diseases or conditions described herein.
- a number of a particular cyotkine's comprising immunogens may be used to produce antibodies specifically reactive with that particular cytokine biomarker.
- a recombinant IL-6 or MIP- l ⁇ or MCP-I or IFN ⁇ or an antigenic fragment thereof can be isolated using methods well known to those of skill in the art.
- Recombinant protein can be expressed in eukaryotic or prokaryotic cells.
- Recombinant protein is the typically used immunogen for the production of monoclonal or polyclonal antibodies.
- a synthetic peptide derived from the known sequences of the cytokine biomarkers and conjugated to a carrier protein can be used an immunogen.
- Naturally occurring protein may also be used either in pure or impure form.
- the product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
- each specific cytokine biomarker can be detected by a variety of immunoassay methods.
- immunoassay methods see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991).
- the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.
- Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case IL-6 or IFN ⁇ or MIP- l ⁇ or MCP-I or antigenic subsequence thereof).
- a protein or antigen of choice in this case IL-6 or IFN ⁇ or MIP- l ⁇ or MCP-I or antigenic subsequence thereof.
- the antibody ⁇ e.g., anti- IL-6, or anti-MIP-1 ⁇ or anti-MCP-1 or anti-IFN ⁇
- the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
- Specific binding of a cytokine to an antibody may typically require an antibody that is selected for its specificity for a particular protein.
- polyclonal antibodies raised to a particular cytokine e.g., IL-6 or MIP-I ⁇ or MCP-I or IFN ⁇
- polyclonal antibodies raised to a particular cytokine can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with a particular cytokine (e.g., IL-6 or MIP- l ⁇ or MCP-I or IFN ⁇ ), and not with other proteins, except for polymorphic variants, orthologs, and alleles of the particular cytokine. This selection may be achieved by subtracting out antibodies which react with the cytokine of interest.
- immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
- solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein ⁇ see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
- the signal of a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
- Antibodies that react only with a particular cytokine ortholog e.g., from specific species such as rat, mouse, or human, can also be detected as described above, by subtracting out antibodies that bind to the same cytokine from another species.
- Immunoassays also often use a labeling agent to specifically bind to and allow for the detection of the complex formed by the antibody and antigen.
- the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
- the labeling agent may be a labeled anti-IL-6 or anti-MIPl ⁇ or anti-MCP-1 or anti-IFN ⁇ antibody.
- the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/ cytokine complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
- Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent.
- the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin.
- detectable moieties are well known to those skilled in the art.
- Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C. In some embodiments, the immunological assay is instantaneous and a read-out for the presence or levels of the cytokine biomarkers is available nearly immediately upon extracting the sample from the acutely painful joint and performing the immunoassay.
- Immunoassays for detecting the cytokine biomarkers in samples may be either competitive or noncompetitive.
- Noncompetitive immunoassays are assays in which the amount of antigen is directly measured.
- the anti-IL-6 or anti-MIPl ⁇ or anti-MCP-1 or anti-IFN ⁇ antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture the corresponding cytokine present in the test sample. The cytokine is thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label.
- the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
- the second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.
- the amount of cytokine biomarker e.g., IL-6 (IL-6 is used as an illustrative example in this description, a similar scenario can be applied to any of the other cytokine biomarkers) present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) cytokine displaced (competed away) from an anti-IL-6 antibody by the unknown IL-6 present in a sample.
- a known amount of IL-6 is added to a sample and the sample is then contacted with an antibody that specifically binds to the IL-6.
- the amount of exogenous IL-6 bound to the antibody is inversely proportional to the concentration of IL-6 present in the sample.
- the antibody is immobilized on a solid substrate.
- the amount of IL-6 bound to the antibody may be determined either by measuring the amount of IL-6 present in a IL- 6/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein.
- the amount of IL-6 may be detected by providing a labeled IL-6 molecule.
- a hapten inhibition assay is another competitive assay.
- the known cytokine e.g., IFN ⁇
- IFN ⁇ is used for purposes of illustration of how a hapten inhibition assay works, any of the other cytokines can be similarly used in such an assay
- a known amount of anti-IFN ⁇ antibody is added to the sample, and the sample is then contacted with the immobilized IFN ⁇ .
- the amount of anti-IFN ⁇ antibody bound to the known immobilized IFN ⁇ is inversely proportional to the amount of IFN ⁇ present in the sample.
- the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
- LOA liposome immunoassays
- the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
- the detectable group can be any material having a detectable physical or chemical property.
- Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
- a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, radiographic, electrical, optical or chemical means.
- Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
- magnetic beads e.g., DYNABEADSTM
- fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
- radiolabels e.g., 3 H, 125 1, 35 S, 14 C, or 32 P
- enzymes e.g., horse radish per
- the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
- Non-radioactive labels are often attached by indirect means.
- a ligand molecule e.g., biotin
- the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- the ligands and their targets can be used in any suitable combination with antibodies that recognize the cytokine biomarkers, or secondary antibodies that recognize the antibodies to the cytokine biomakers.
- the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
- Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases.
- Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
- Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
- Means of detecting labels are well known to those of skill in the art.
- means for detection include a scintillation counter or photographic film as in autoradiography.
- the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
- CCDs charge coupled devices
- enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
- simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
- agglutination assays can be used to detect the presence of the target antibodies.
- antigen-coated particles are agglutinated by samples comprising the target antibodies.
- none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
- Detection methods employing immunoassays are particularly suitable for practice at the point of patient care. Such methods allow for immediate diagnosis and/or prognostic evaluation of the patient.
- Point of care diagnostic systems are described, e.g., in U.S. Patent 6,267,722 which is incorporated herein by reference.
- Other immunoassay formats are also available such that an evaluation of the biological sample can be performed without having to send the sample to a laboratory for evaluation.
- these assays are formatted as solid assays where a reagent, e.g., an antibody is used to detect the cytokine.
- Exemplary test devices suitable for use with immunoassays such as assays of the present invention are described, for example, in U.S. Patents 7,189,522; 6,818,455 and 6,656,745.
- the present invention provides methods for detection of polynucleotide sequences which code for the cytokine biomarkers (e.g., IL-6, MIP-I ⁇ , MCPl and IFN ⁇ ) in a biological sample, e.g., for the diagnosis of acute joint injury.
- a biological sample refers to a cell or population of cells or a quantity of tissue or fluid from a patient. Most often, the sample has been removed from a patient, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, i.e., without removal from the patient. Typically, a "biological sample” will contain cells from the patient, but the term can also refer to noncellular biological material, such as noncellular fractions of the fluid from a potentially affected joint.
- amplification-based assays are used to measure the level of IL- 6, or MlP-l ⁇ , or MCPl, or IFN ⁇ .
- the IL-6, or MlP-l ⁇ , or MCPl, or IFN ⁇ nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR).
- an amplification reaction e.g., Polymerase Chain Reaction, or PCR.
- the amount of amplification product will be proportional to the amount of template in the original sample.
- Comparison to appropriate controls provides a measure of the copy number of the cytokine biomarker associated gene. Methods of quantitative amplification are well known to those of skill in the art.
- RT-PCR methods are well known to those of skill (see, e.g., Ausubel et ah, supra).
- quantitative RT-PCR e.g., a TaqMan® assay, is used, thereby allowing the comparison of the level of mRNA in a sample with a control sample or value.
- the known nucleic acid sequences for IL-6, MlP-l ⁇ , MCPland IFN ⁇ are sufficient to enable one of skill to routinely select primers to amplify any portion of the gene.
- Suitable primers for amplification of specific sequences can be designed using principles well known in the art (see, e.g., Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995)).
- a TaqMan® based assay is used to quantify the cytokine biomaker-associated polynucleotides.
- TaqMan® based assays use a fiuorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end.
- the 5' nuclease activity of the polymerase e.g., AmpliTaq®, results in the cleavage of the TaqMan® probe.
- This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, literature provided by Perkin-Elmer, e.g., www2.perkin-elmer.com).
- hybridization based assays can be used to detect the amount of IL-6, or MIP-I ⁇ , or MCPl, or IFN ⁇ in the cells of a biological sample.
- assays include dot blot analysis of RNA as well as other assays, e.g., fluorescent in situ hybridization, which is performed on samples that comprise cells.
- Other hybridization assays are readily available in the art.
- the present methods can be used in the diagnosis, prognosis and treatment of acute joint inflammation.
- the acute inflammation is in the knee, shoulder, elbow, wrist or ankle.
- Acute inflammation of the joint at any stage during the 30 week period from the onset of pain can be diagnosed using the methods of the present invention.
- the level and/or presence of IL-6 or MIP-I ⁇ or MCP-I or IFN ⁇ polynucleotide or polypeptide will be detected in a biological sample, thereby detecting the presence or absence of acute inflammation in the biological sample.
- the biological sample will comprise a tissue sample from a tissue suspected of being subject to acute inflammation, e.g., a fluid sample or lavasate of a joint.
- a joint sample determined to contain the cytokine biomarkers of the present invention e.g., a knee fluid sample shown to contain IL-6 and or MCP-I or a knee sample shown to contain IL-6 and MCP-I and MIP- l ⁇ or a knee sample shown to contain IL-6 and MCP-I and IFN ⁇
- a knee fluid sample shown to contain IL-6 and or MCP-I or a knee sample shown to contain IL-6 and MCP-I and MIP- l ⁇ or a knee sample shown to contain IL-6 and MCP-I and IFN ⁇ will be analyzed for IL-6, MCP-I, MIP l ⁇ and/or IFN ⁇ levels to further characterize the injured joint, e.g., the efficacy of certain treatments.
- the amount of IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ polynucleotide or polypeptide that will indicate the presence of a acute inflammation and classify the patient as candidate for treatment will depend on numerous factors, including the affected joint, the age, sex, medical history, etc., of the patient, the cell type, the assay format, etc.
- a level of IL-6, MCP- 1 , IFN ⁇ and/or MIP- 1 ⁇ in a biological sample will not be quantified or directly compared with a control sample, but will rather be detected relative to a "diagnostic presence" of IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ wherein a "diagnostic presence” refers to an amount of IL-6, MCP-I , IFN ⁇ and/or MIP-I ⁇ polynucleotide or polypeptide that indicates the presence or likelihood of acute inflammation in the joint of the mammal from which the sample was taken.
- a "diagnostic presence” will be detectable in a simple assay giving a positive or negative result, where a positive “detection” of a "diagnostic presence” of IL-6, MCP-I, IFN ⁇ and/or MIP- l ⁇ polynucleotide or polypeptide indicates the presence of acute joint inflammation in the mammal.
- the IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ level need not be quantified for a "diagnostic presence" to be detected. Rather any method of determining whether IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ is present at levels higher than in a normal or control, pain- free joint, sample, or patient may be used.
- a "diagnostic presence” does not refer to any absolute quantity of IL-6, MCP-I, IFN ⁇ and/or MIP- l ⁇ , but rather on an amount that, depending on the biological sample, assay conditions, medical condition of the mammal, etc., is sufficient to distinguish the level in an acutely inflamed joint sample from a joint sample from a normal or control patient.
- Such methods can be practiced regardless of whether any IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ polynucleotide or polypeptide is normally present, or "expected" to be present, in a particular control sample.
- IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ may not be expressed in certain normal joints, resulting in a complete absence of IL-6, MCP-I, IFN ⁇ and/or MIP- l ⁇ in a control biological sample from such a joint.
- a "diagnostic presence” refers to any detectable amount of IL-6, MCP-I, IFN ⁇ and/or MIP- 1 ⁇ , using any assay.
- a "diagnostic presence" represents a level that is higher than the normal level, preferably representing a "statistically significant” increase over the normal level.
- a "diagnostic presence" of IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ polynucleotide, polypeptide, and/or protein activity in a biological sample will be at least about 1.5, 2, 5, 10,100, 200, 500, 1000 or more fold greater than a level expected in a sample taken from a normal patient or from the normal contralateral joint of the same patient.
- the "diagnostic presence" of IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ polynucleotide, polypeptide, and/or protein activity in a biological sample will be dependent on the specific type of joint. For example, when evaluating the diagnostic presence of a cytokine in the knee vs. the diagnostic presence of a cytokine in the shoulder different levels of the particular cytokine biomarker may be indicative of an acutely inflamed joint, hi some embodiments, the diagnostic presence of the cytokine biomarker will depend on the age of the patient.
- the difference in cytokine biomarker levels between the control knee and the affected knee in the older patient population will be at least about 1.5, 2, 5, 10,100, 200, 500, 1000 fold, hi some embodiments other diagnostic methods including radiograph can be combined with the methods of the present invention to diagnose acute inflammation in a joint.
- the presence or level of IL-6, MCP- 1 , IFN ⁇ and/or MIP- 1 ⁇ or polypeptide fragments thereof can be used to designate a patient as candidate for treatment.
- the type of treatment e.g., anti-inflammatory agent or surgery, can be then tailored to severity of the condition as determined by the presence or level of the cytokine biomarkers.
- the present methods can also be used to assess the efficacy of a course of treatment.
- a mammal with acute joint injury found to contain an elevated amount of IL- 6, MCP-I, IFN ⁇ and/or MIP- l ⁇ polynucleotide or polypeptide or fragment thereof the efficacy of an anti-inflammatory treatment can be assessed by monitoring, over time, IL-6, MCP-I, IFN ⁇ and/or MIP- l ⁇ levels.
- the methods detecting acute joint inflammation can comprise the detection of one or more cytokine biomarker polynucleotide, polypeptide or polypeptide fragment sequences. Accordingly, IL-6, MCP-I, IFN ⁇ and/or MIP- l ⁇ can be used either alone or in any combination for the diagnosis or prognosis acute joint inflammation.
- a patient can be selected as a candidate for treatment based on determining the presence of the inflammatory cytokine in a biological sample such as blood, serum, plasma, or the like.
- a biological sample such as blood, serum, plasma, or the like.
- the blood (or serum or plasma) sample can be analyzed using an immunoassay or, in some embodiments, using an amplification reactions such as PCR.
- a cytokine biomarker e.g., IL-6, MCP-I , IFN ⁇ and/or MIP-I ⁇
- imaging methodology refers to a procedure or modality for generating an image of a detectable moiety in vivo, ex vivo, or in vitro.
- imaging methods include radioscintigraphy, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging methods, nuclear magnetic resonance, fluorescence detection, ultra-sound, and chemiluminescent detection (see, e.g., Dahnhert, Radiology Review Manual (4th ed.
- In vivo imaging is used to visualize the presence of the cytokine biomarker in the joint of a patient as described herein.
- radiographic imaging methods are known in the art.
- an antibody to IL-6, MCP-I, IFN ⁇ and/or MIP- l ⁇ is linked to a moiety for detection.
- Useful agents include, but are not limited to, radioisotopes, dyes, contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI).
- MRI magnetic resonance imaging
- In vivo imaging methods include radionuclide imaging, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging method, fluorescence detection, and chemiluminescent detection. In vivo imaging is used to visualize the presence of the cytokine biomarker
- antibodies to the cytokine biomarker are radioactively labeled.
- the use of radio-labelled antibodies for in vivo diagnosis is 1 known in the art.
- the label used will depend on the imaging modality chosen.
- radioactive labels such as Indium- 111, Technetium-99m, or Iodine- 131 can be used for planar scans or single photon emission computed tomography (SPECT).
- Positron emitting labels such as Fluorine- 19 can also be used for positron emission tomography (PET).
- paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used.
- Radioactive metals for labeling often have short half lives, for example, ranging from 1 hour to 3.5 days.
- Radioagents such as scandium-47, gallium-67, gallium-68, technetiium-99m, and indium- 111, are often used for gamma camera imaging, gallium-68 is often used for positron emission tomography.
- U.S. Pat. No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to a MRI enhancing agent.
- a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions.
- Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
- chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
- Chelates can be coupled to the antibodies using standard chemistries.
- the chelate may be linked to the antibody by a group which enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking.
- Other, more unusual, methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659 to Hawthorne, entitled “Antibody Conjugates", issued Apr. 25, 1989.
- useful metal-chelate combinations may include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125 I 3 131 I, 123 I, 124 1, 62 Cu, 64 Cu, 18 F, 111 In 5 - 67 Ga; . 68 Ga, 99m Tc, 94m Tc, 86 Y, I 86 Re, 188 R, 11 C, 13 N, 15 O, and/or 76 Br, for radio-imaging.
- 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125 I 3 131 I, 123 I, 124 1, 62 Cu, 64 Cu, 18 F, 111 In 5 - 67 Ga; . 68 Ga, 99m Tc, 94m Tc, 86 Y, I 86 Re, 188 R
- chelates when complexed with non-radioactive metals, such as manganese, iron and gadolinium may be useful for MRI, when used along with the cytokine biomarker antibodies disclosed herein.
- Macrocyclic chelates such as NOTA, DOT A, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper, respectively.
- metal-chelate complexes can be stabilized by tailoring the ring size to the metal of interest.
- Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, can also be used.
- agents such as particles or liposomes conjugated to an antibody to a cytokine biomarker, can be used as agents for ultrasound imaging.
- Ultrasound enhancing agents are disclosed in United States Patent Applications US20040219203 Al and US20050014207A1.
- gas-filled liposomes may be used. See Maresca, G. et al., Eur J. Radiol. Suppl 2 S171-178 (1998); Demos, Sm. Et al. J. Drug Target 5 507-518 (1998); and Unger, E. et al., Am J. Cardiol. 81 58G-61G (1998).
- a labeled antibody can be administered for in vivo imaging using a variety of techniques.
- the antibody is introduced directly into the joint, e.g., the injured knee or other joint, and the joint is visualized.
- the antibody is administered intravenously and the joint of interest is visualized.
- the patient selected for treatment is treated by surgically correcting the abnormalities in the affected joint.
- treatment methods to ameliorate production of inflammatory cytokines are employed.
- Such treatment methods include, for example, surgical debridement including open or arthroscopic meniscectomy, or labral debridement in the shoulder.
- surgical procedures to remove loose bodies, cartilage or meniscal repair, ligament alignment or synovectomy can be used.
- bony realigning procedures of an extra articular nature that may assist in the removal of eccentric strain and pressure forces on a joint, may be employed.
- treatment agents can be administered to the patient.
- Treatment agents can include, for example, anti-inflammatory treatment therapies.
- Nonsteroidal anti-inflammatory drugs are well known to those of skill in the art.
- the treatment methods can involve an NSAID, e.g., ibuprofen, aspirin or paracetamol.
- NSAID e.g., ibuprofen, aspirin or paracetamol.
- steroids e.g., glucocorticoids, reduce inflammation by binding to Cortisol receptors.
- steroids are used to treat the acutely inflamed joint diagnosed using the methods of the present invention.
- Other treatment regiments can be based on neutralizing the elevated cytokine biomarkers.
- antagonists of one or more of the cytokines from the group of IL- 6, MIP-I ⁇ , MCP-I and IFN- ⁇ can be selected for treatment.
- Antibodies are an example of a suitable antagonists and include mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies or fragments thereof. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to different species (see, e.g., Boyce et al., Annals of Oncology 14:520-535 (2003)).
- variable (V) segments of the genes from a mouse monoclonal antibody may be joined to human constant (C) segments.
- a typical chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector regions from a human antibody.
- Humanized antibodies have variable region framework residues substantially from a human antibody (termed an acceptor antibody) and complementarity determining regions substantially from a mouse-antibody, (referred to as the donor immunoglobulin). See Queen et al., Proc. Natl. Acad. ScL USA 86:10029-10033 (1989) and WO 90/07861, US 5,693,762, US 5,693,761, US 5,585,089, US 5,530,101 and Winter, US 5,225,539. The constant region(s), if present, are also substantially or entirely from a human immunoglobulin.
- Antibodies can be obtained by conventional hybridoma approaches, phage display (see, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047), use of transgenic mice with human immune systems (Lonberg et al., WO93/12227 (1993)), among other sources.
- Nucleic acids encoding immunoglobulin chains can be obtained from hybridomas or cell lines producing antibodies, or based on immunoglobulin nucleic acid or amino acid sequences in the published literature.
- Other antagonists of the biomarker cytokines can also be used for treatment purposes.
- a class of antagonists that can be used for the purposes of the present invention are the soluble forms of the receptors for the cytokine biomarkers.
- an IL-6 antagonist is an anti-IL-6 antibody that specifically binds to IL-6.
- a specific antibody has the ability to inhibit or antagonize the action of IL-6 systemically.
- the antibody binds IL-6 and prevents it from interacting with or activating its receptors (e.g.. IL-6R ⁇ or IL-6R ⁇ ).
- the activity of IL-6 can be antagonized by using an antagonist to the interleukin-6 receptors (IL-6R).
- IL-6R interleukin-6 receptors
- U.S. Application number 2006251653 describes methods for treating interleukin-6 related disease and discloses a number of interleukin-6 antagonists including, for example, humanized anti-IL-6R antibodies and chimeric anti-IL-6R antibodies.
- an IL-6 or IL-6R derivative can be used to block and antagonize the interaction between IL- 6/IL-6R.
- MCPl antagonists include anti-MCPl antibodies.
- Antibodies to MCPl can function by disrupting the ability of MCPl to bind its binding partners.
- Binding partners of MCPl include CD234, matrix metalloproteinase 1, matrix metalloproteinase 3, matrix metalloproteinase 8, a number of CC chemokine receptors including, e.g., CC chemokine receptor 2 and CC chemokine receptor 5.
- WO05018431 describes a variety of ways to disrupt the interaction between MCPl and its binding partners, for instance, by using antibodies, or an MCPl derivative. Alternatively chemical compositions that antagonize the activity of MCPl can be used. U.S. Application 2003096705 describes compounds which antagonize MCPl function. In other embodiments, agents that bind to MCPl binding partners, e.g., antibodies to MCPl binding partners can be used to block the interaction between MCPl and its binding partner and inhibit activity.
- IFN ⁇ antagonists for use in the invention are antibodies that bind IFN ⁇ antibodies, e.g., fontolizumab. Such antibodies can be neutralizing antibodies that block IFN ⁇ activity.
- Humanized antibodies that bind to IFN ⁇ are described, e.g., in U.S. Patent No. 6,329,511 and U.S. Patent No. 7,183,390.
- the antibody indirectly inhibits interferon gamma.
- IFN ⁇ binding partner include, for example, interferon gamma receptor 1 , interferon gamma receptor 2, TNF ⁇ and protein disulfide isomerase A3.
- Antibodies to IFN ⁇ 's binding partners can also be used to inhibit the activity of IFNg and thus serve as antagonists of IFN ⁇ .
- Other interferon antagonists useful for the treatment of interferon-related related diseases are described, for example, in U.S. Application 2003138404.
- MIPl ⁇ can be antagonized, for example, through the use of anti- MIP 1 ⁇ antibodies. Either blocking MIP- 1 ⁇ or one of its binding partners can function to prevent the activity of MIPl ⁇ .
- Known binding partners of MIPl ⁇ are, for example, CC chemokine receptor 3, CC chemokine receptor 5 and CC chemokine receptor 8. Fragments of MIPl ⁇ or any of its CC chemokine receptor binding partners can also be used to block productive interaction and thus inhibit or antagonize the activity of MIPl ⁇ .
- Inhibitors of IL-6, MCP-I, IFN ⁇ and/or MIP- l ⁇ can be administered to a patient for the treatment of acute joint pain. As described in detail below, the inhibitors are administered in any suitable manner, optionally with pharmaceutically acceptable carriers.
- the identified inhibitors can be administered to a patient at therapeutically effective doses to treat acute joint injury or prevent further deterioration of the joint due to acute inflammation.
- the compounds are administered to a patient in an amount sufficient to elicit an effective protective or therapeutic response in the patient.
- An effective therapeutic response is a response that at least partially arrests or slows the symptoms or complications of the disease.
- An amount adequate to accomplish this is defined as "therapeutically effective dose.”
- the dose will be determined by the efficacy of the particular IL-6, MCP-I, IFN ⁇ and/or MIP-I ⁇ inhibitors employed and the condition of the subject, as well as the body weight or surface area of the area to be treated.
- the size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound or vector in a particular subject.
- Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (the dose lethal to 50% of the population) and the ED 5O (the dose therapeutically effective in 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD 50 /ED5 0 .
- Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to normal cells and, thereby, reduce side effects.
- the data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage can vary within this range depending upon the dosage form employed and the route of administration.
- the therapeutically effective dose can be estimated initially from cell culture assays.
- a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
- IC 50 the concentration of the test compound that achieves a half-maximal inhibition of symptoms
- levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC).
- HPLC high performance liquid chromatography
- the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.
- compositions for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients.
- the compounds and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including orally and by direct injection into the affected joint.
- Formulations for the forms of administration suitable with the methods of the present invention are well known to those of skill in the art.
- the compounds can be formulated for administration by injection, for example, by bolus injection.
- Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative.
- the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents, for example, suspending, stabilizing, and/or dispersing agents.
- the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen- free water, before use. VII. Kits for Use in Diagnostic and/or Prognostic Applications
- kits for use in diagnostic, research, and therapeutic applications as described herein.
- such kits may include any or all of the following: assay reagents, buffers, cytokine biomarker- specific nucleic acids or antibodies, hybridization probes and/or primers.
- kits of the present invention include selective binding partners for two of the four cytokine biomarkers of the present invention including IL-6. In some embodiments, the kits of the present invention include selective binding partners for three of the four cytokine biomarkers of the present invention, including IL-6. hi some embodiments, the kits of the present invention include selective binding partners for two of the four cytokine biomarkers of the present invention including MCP-I . In some embodiments, the kits of the present invention include selective binding partners for three of the four cytokine biomarkers of the present invention, including MCP-I. In some embodiments, the kit contains all four of the cytokine biomarkers.
- kits of the present invention include the selective binding partners on a continuous solid surface. In such embodiments, the detection of presence or level of all the cytokine biomarkers can be assessed simultaneously. In some embodiments, the binding partners for the cytokine biomarkers of the present invention are present on individual solid surfaces. In such embodiments the presence and level of the cytokine biomarkers can be detected simultaneously or sequentially.
- the selective binding partners are antibodies to two or more of the cytokines: IL-6, MlPl ⁇ , IFN ⁇ or MCPl.
- detection of the presence or level of the cytokine biomarker is by immunoassay, hi some embodiments, the kits include primers specific to amplifying any two or more of the four cytokine biomarkers of the present invention.
- primers specific to amplifying any two or more of the four cytokine biomarkers of the present invention Those of skill in the art can easily determine how to design primers specific for amplifying a particular cytokine biomarker based on the biomarker nucleotide sequence. Nucleotide detection methods can be used with kits comprising primers.
- polymerase chain reactions are used to detect the cytokine biomarkers.
- the polymerase chain reaction is RT-PCR.
- kits a device to be utilized for the extraction of the biological sample is also included in the kit.
- the extraction device e.g., a syringe and a needle, or a catheter
- the kit can directly extract the biological sample from the potentially affected joint into a chamber containing the selective binding partners for the cytokine biomarkers.
- the kit thus allows for immediate assessment of the cytokine biomarkers' presence and/or level and, therefore, immediate diagnosis of a patient suffering from nonimmune inflammatory acute joint injury.
- kits are particularly suitable for use at the point of care.
- An example of a point of care diagnostic system is described in U.S. Patent 6,267,722 which is incorporated herein by reference.
- Other devices whose design can be adapted for use with the kits of the present invention are described, for example, in U.S. Patent numbers 7,198,522 and 6,818,455.
- the potentially affected joint does not contain sufficient fluid for extraction.
- a lavage of the potentially acutely injured joint is necessary.
- the kit may include a solution to be used for the extraction of the biological sample from the joint.
- This solution included in the kit can be, for example, a physiologic solution, e.g. saline.
- the kit comprises a material, e.g., an absorbent material, that can be introduced into the joint to obtain the biological sample.
- the absorbent material can comprise material such as cotton and/or a sponge to obtain the biological sample.
- the kit comprises a needle or wire or device, such as a strip, that can be introduced into the joint where the need or wire also includes a solid surface to which an antibody to an inflammatory cytokine is attached.
- the solid surface can be any surface suitable for introduction into a joint.
- kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. VIII. Examples
- Example 1- Subjects and knee lavage
- Inclusion criteria Patients 18-80 years old presenting with symptoms of knee pain, either secondary to arthritis or a traumatic event and who satisfied clinical indications for arthroscopic surgery following MRI of the symptomatic knee.
- Exclusion criteria less than 18 years old, recent (within 3 months) intra-articular corticosteroid injection, past or current medical history of autoimmune disease. In addition, no patients involved in a worker's compensation claim or personal injury litigation, were enrolled in the study.
- Example 4 Patient pain scores, age and gender profile by group - knee samples
- the mean ( ⁇ SEM) self-reported visual analog score (VAS) for the normal control group, operative knee group and non-operative knee group was O ⁇ O, 1.6 ⁇ 0.4 and 6.4 ⁇ 0.2, respectively.
- the mean age of the normal control group, operative knee group and non- operative knee group was 45.7 ⁇ 1.7, 51.5 ⁇ 2.9 and 52. l ⁇ 3.3 years, respectively.
- the ratio of male:female of the normal control group, operative knee group and non-operative knee group was 8:6, 26:9 and 18:8, respectively.
- Table 1 illustrates levels of cytokines from a cohort of normal patients whose knees were not painful.
- Table 2 illustrates cytokine biomarker levels from patients who experienced various levels of pain (as indicated by the VAS score) in their knee and were selected as candidates for surgery
- Patient B209 had a traumatic shoulder dislocation with a large effusion.
- the joint was aspirated and synovial fluid was removed for analysis.
- the sample was handled in the same manner as the knee samples described above.
- Patient 312 had a rotator cuff injury with a cartilage lesion of the labrum, called a SLAP lesion.
- the joint could not be aspirated, so approximately 20 cc of normal saline was injected into the shoulder.
- the lavage procedure was performed similarly to the lavages described for the normal knees.
- Example 6 Using IL-6 as optimal specificity biomarker of acute joint injury
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US20050152905A1 (en) * | 2002-08-22 | 2005-07-14 | Omoigui Osemwota S. | Method of biochemical treatment of persistent pain |
US20070122405A1 (en) * | 2004-03-12 | 2007-05-31 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
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US5866007A (en) * | 1994-05-19 | 1999-02-02 | The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration | Method and apparatus for the collection, storage, and real time analysis of blood and other bodily fluids |
US20050152905A1 (en) * | 2002-08-22 | 2005-07-14 | Omoigui Osemwota S. | Method of biochemical treatment of persistent pain |
US20070122405A1 (en) * | 2004-03-12 | 2007-05-31 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
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