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WO2005056753A2 - Procedes de determination de la classification de l'arthrite juvenile - Google Patents

Procedes de determination de la classification de l'arthrite juvenile Download PDF

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Publication number
WO2005056753A2
WO2005056753A2 PCT/US2004/035255 US2004035255W WO2005056753A2 WO 2005056753 A2 WO2005056753 A2 WO 2005056753A2 US 2004035255 W US2004035255 W US 2004035255W WO 2005056753 A2 WO2005056753 A2 WO 2005056753A2
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interest
nucleotide sequence
monocyte sample
subject
peripheral blood
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WO2005056753A3 (fr
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David Glass
Michael Barnes
Susan Thompson
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Cincinnati Childrens Hospital Medical Center
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Cincinnati Childrens Hospital Medical Center
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Priority claimed from US10/970,698 external-priority patent/US20050164244A1/en
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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
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of differential gene expression in juvenile arthritis.
  • BACKGROUND OF THE INVENTION Chronic inflammatory arthritis is a source of morbidity for about 70,000 children in the United States alone.
  • There are currently three classification systems for the juvenile arthritides Petty, R.E. and Cassidy, J.T. (2001) The Juvenile Idiopathic Arthritides. In Textbook ofPediatric Rheumatology. 4 th ed. J. T. Cassidy and R.E. Petty, ed. W.B. Sauders Co., St. Louis p. 214, herein inco ⁇ orated by reference).
  • Juvenile rheumatoid arthritis is defined by American College of Rheumatology criteria, while juvenile chronic arthritis (JCA) and juvenile idiopathic arthritis (JIA) correspond to European League against rheumatism and the international League of Associations for Rheumatology criteria, respectively. See Petty, R.E. and Cassidy, J.T. (2001) The Juvenile Idiopathic Arthritides. In Textbook ofPediatric Rheumatology. 4 th ed. J. T. Cassidy and R.E. Petty, ed. W.B. Sauders Co., St. Louis p. 214; Cassidy et al. (1986) Arthritis Rheum.
  • Spondyloarthropathies although more common in adults, can begin during childhood and may be confused with late-onset pauciarticular JRA at onset when there is an absence of enthesitis and axial involvement. The presence of enthesitis is a useful predictor for juvenile onset spondyloarthropathy (JSpA), and HLA-B27 is frequently positive.
  • JSpA juvenile onset spondyloarthropathy
  • Cytokines are a large group of polypeptides and small proteins that are secreted by cells of the immune system. Although cytokine functions are complex, cytokine profiles are relevant parameters of an immune response. The ratio of pro- and anti-inflammatory cytokines and the T helper cell subtypes is considered important in the pathogenesis of autoimmune diseases including juvenile idiopathic arthritis. The measurement of cytokines and chemotactic cytokines in body fluids and synovia! tissue has provided insight into the type of immune and inflammatory reaction.
  • Methods and kits for determining disease classification, particularly of juvenile arthritides, and for analyzing disease progression in subjects exhibiting juvenile arthritis are provided.
  • the inventions are based on the novel discovery that certain inflammatory related nucleotide sequences are expressed differently in the various classifications of juvenile arthritis, particularly pauciarticular juvenile arthritis, polyarticular juvenile arthritis, systemic onset juvenile rheumatoid arthritis, and juvenile onset spondyloarthropathies.
  • the expression patterns of the nucleotide sequences of interest in peripheral blood monocytes and/or synovial fluid monocytes differ among the categories of juvenile arthritides.
  • Methods of the invention allow determination of disease classification by analyzing the expression patterns of the nucleotide sequences of interest from various tissues in a subject.
  • the invention further provides a method of analyzing disease progression and kits for performing the methods of the invention. Additionally, the invention provides methods of identifying expression modulating compounds and arthritis modulating compounds.
  • the present invention involves analyzing the expression pattern of CXCL chemokines (composed of both angiogenic and angiostatic factors) to classify juvenile arthritis in a subject, to predict the course of the juvenile arthritis, and/or to predict the efficacy of treatments.
  • the invention is based on the discovery that certain chemokines are differentially expressed in peripheral blood monocytes (PBMC) and synovial fluid monocytes (SFMC) in various classifications of juvenile rheumatoid arthritis.
  • PBMC peripheral blood monocytes
  • SFMC synovial fluid monocytes
  • chemokines are members of a family of angiogenic and angiostatic cytokines defined by the presence or absence of an ELR amino acid motif.
  • Gene expression analysis of peripheral blood monocytes identified angiogenic chemokines including, but not limited to, CXCL1, CXCL2, CXCL3, and CXCL8, with altered expression in polyarticular samples compared to other disease subtypes or controls. Expression of these and additional angiogenic cytokines in synovial fluid monocytes were equivalent between juvenile arthritis types.
  • the invention provides a method of determining disease classification in a subject.
  • the method involves the steps of obtaining a peripheral blood monocyte sample, a synovial fluid monocyte sample, or both a peripheral blood monocyte sample and a synovial fluid monocyte sample, from a subject, assaying the expression level of a nucleotide sequence of interest in the samples, and comparing the expression levels of the nucleotide sequence of interest to a standard expression pattern to determine disease classification.
  • the method further comprises isolating RNA from the samples.
  • Nucleotide sequences of interest include, but are not limited to, the nucleotide sequences set forth in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55; nucleotide sequences encoding the amino acid sequences set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 56, and fragments and variants thereof. Many of the nucleotide sequences of interest are CXCL chemokines.
  • the expression level assay analyzes the polypeptide encoded by the nucleotide sequence of interest.
  • the subject is a mammal, particularly a human.
  • the subject exhibits a juvenile arthritis.
  • the disease classification is classification of a juvenile arthritis including but not limited to, pauciarticular juvenile arthritis, polyarticular juvenile arthritis, systemic onset juvenile rheumatoid arthritis, and juvenile onset spondyloarthropathy.
  • the invention provides a method of determining juvenile arthritis classification in a subject exhibiting juvenile arthritis.
  • the method involves the steps of obtaining a peripheral blood monocyte sample, a synovial fluid monocyte sample, or both, from a subject, assaying the expression level of a nucleotide sequence of interest in the samples, and comparing the expression levels of the nucleotide sequence of interest to a standard expression pattern to determine disease classification.
  • Nucleotide sequences of interest are described elsewhere herein.
  • expression levels of multiple nucleotide sequences of interest are compared to a standard expression pattern.
  • expression levels of at least five, at least ten, at least fifteen, at least eighteen, or at least twenty nucleotide sequences of interest are compared to a standard expression pattern.
  • kits for performing the method comprising a peripheral blood monocyte sample collection reagent, a synovial fluid sample collection reagent, and a detection reagent for at least one nucleotide sequence of interest.
  • the kit comprises detection reagents for at least 18 nucleotide sequences of interest.
  • the invention provides a method of analyzing disease progression in a subject exhibiting juvenile arthritis.
  • the method involves obtaining a first peripheral blood monocyte sample, a first synovial fluid mononuclear cell sample, or both, from the subject, assaying a first expression level of a nucleotide sequence of interest in the first samples, obtaining a second peripheral blood monocyte sample, a second synovial fluid monocyte sample, or both a second peripheral blood monocyte sample and a second synovial fluid monocyte sample, from the subject, assaying a second expression level of a nucleotide sequence of interest in the second samples, and comparing the first and second expression levels of the nucleotide sequence of interest.
  • the invention further provides a kit for performing the method of the invention.
  • the invention provides a method of identifying a nucleotide sequence of interest expression modulating compound.
  • the method involves obtaining a first peripheral blood monocyte sample, a first synovial fluid monocyte sample, or both from a subject exhibiting juvenile arthritis, assaying a first expression level of a nucleotide sequence of interest in the first samples, administering a compound of interest to the subject, obtaining a second peripheral blood monocyte sample, a second synovial fluid monocyte sample, or both from the subject, assaying a second expression level of a nucleotide sequence of interest in the subject, and comparing the first and second expression levels of the nucleotide sequence of interest.
  • the subject is a human, mouse, rabbit, dog, pig, goat, cow, rat, monkey, chimpanzee, or sheep.
  • the compound of interest is administered to a subject, cells obtained from a subject, or cells cultured from a subject.
  • the invention provides a method of identifying an arthritis modulating compound.
  • the method involves obtaining a first peripheral blood monocyte sample and a first synovial fluid monocyte sample from a subject exhibiting juvenile arthritis, assaying a first expression level of a nucleotide sequence of interest in the first samples, administering a compound of interest to the subject, obtaining a second peripheral blood monocyte sample and a second synovial fluid monocyte sample from the subject, assaying a second expression level of a nucleotide sequence of interest in the subject, and comparing the first and second expression levels of the nucleotide sequence of interest.
  • the compound of interest is administered to a subject, cells obtained from a subject, or cells cultured from a subject.
  • the invention further provides an arthritis modulating compound identified by the method.
  • the invention provides a method of determining juvenile arthritis classification.
  • the method involves the steps of obtaining one or more biological samples from the subject and assaying the expression pattern of nucleotide sequences of interest such as, but not limited to, CXCL chemokines in the biological samples to determine juvenile arthritis classification.
  • the biological sample is a peripheral blood monocyte sample.
  • the biological sample is a synovial fluid monocyte sample.
  • multiple biological samples, such as a peripheral blood monocyte sample and a synovial fluid monocyte sample are obtained. Multiple biological samples may be obtained at multiple time points.
  • the invention provides a method of determining the extent of juvenile arthritis progression in a subject exhibiting juvenile arthritis.
  • the method involves the steps of obtaining one or more biological samples from the subject and assaying the expression pattern of nucleotide sequences of interest such as, but not limited to, CXCL chemokines in the biological samples to determine juvenile arthritis classification.
  • the biological sample is a peripheral blood monocyte sample.
  • the biological sample is a synovial fluid monocyte sample.
  • multiple biological samples, such as a peripheral blood monocyte sample and a synovial fluid monocyte sample, are obtained. Multiple biological samples may be obtained at multiple time points.
  • the invention provides a method of determining disease classification in a subject.
  • the method involves the steps of obtaining a peripheral blood monocyte sample from the subject, assaying the expression level of a nucleotide sequence of interest in the sample, and comparing the nucleotide sequence of interest expression level to a standard expression pattern to determine disease classification.
  • the invention provides a method of determining disease classification in a subject. The method involves the steps of obtaining a synovial fluid monocyte sample from the subject, assaying the expression level of a nucleotide sequence of interest in the sample, and comparing the nucleotide sequence of interest expression level to a standard expression pattern to determine disease classification.
  • the invention provides a method of determining a juvenile arthritis classification in a subject exhibiting juvenile arthritis.
  • the method involves the steps of obtaining a peripheral blood monocyte sample from the subject, assaying the expression level of a nucleotide sequence of interest in the sample, and comparing the nucleotide sequence of interest expression level to a standard expression pattern to determine disease classification.
  • the invention provides a kit for performing the method comprising a peripheral blood monocyte sample collection reagent and a detection reagent for a nucleotide sequence of interest.
  • the invention provides a method of determining a juvenile arthritis classification in a subject exhibiting juvenile arthritis.
  • the method involves the steps of obtaining a synovial fluid monocyte sample from the subject, assaying the expression level of a nucleotide sequence of interest in the sample, and comparing the nucleotide sequence of interest expression level to a standard expression pattern to determine disease classification.
  • the invention provides a kit for performing the method comprising a synovial fluid monocyte sample collection reagent and a detection reagent for a nucleotide sequence of interest.
  • the invention provides a method of analyzing disease progression in a subject exhibiting juvenile arthritis.
  • the method involves the steps of obtaining a first peripheral blood monocyte sample from the subject, assaying a first expression level of a nucleotide sequence of interest in the sample, obtaining a second peripheral blood monocyte sample from the subject, assaying a second expression level of the nucleotide sequence of interest, and comparing the first and second expression levels of the nucleotide sequences of interest.
  • the invention provides a method of analyzing disease progression in a subject exhibiting juvenile arthritis.
  • the method involves the steps of obtaining a first synovial fluid monocyte sample from the subject, assaying a first expression level of a nucleotide sequence of interest in the sample, obtaining a second synovial fluid monocyte sample from the subject, assaying a second expression level of the nucleotide sequence of interest, and comparing the first and second expression levels of the nucleotide sequences of interest.
  • the invention provides a method of identifying a nucleotide sequence of interest expression modulating compound.
  • the method involves the steps of obtaining a first peripheral blood monocyte sample from the subject, assaying a first expression level of a nucleotide sequence of interest in the sample, administering a compound of interest, obtaining a second peripheral blood monocyte sample from the subject, assaying a second expression level of the nucleotide sequence of interest, and comparing the first and second expression levels of the nucleotide sequences of interest.
  • the invention provides a method of identifying a nucleotide sequence of interest expression modulating compound.
  • the method involves the steps of obtaining a first synovial fluid monocyte sample from the subject, assaying a first expression level of a nucleotide sequence of interest in the sample, administering a compound of interest, obtaining a second synovial fluid monocyte sample from the subject, assaying a second expression level of the nucleotide sequence of interest, and comparing the first and second expression levels of the nucleotide sequences of interest.
  • the invention provides a method of identifying an arthritis modulating compound.
  • the method involves the steps of obtaining a first peripheral blood monocyte sample from the subject, assaying a first expression level of a nucleotide sequence of interest in the sample, administering a compound of interest, obtaining a second peripheral blood monocyte sample from the subject, assaying a second expression level of the nucleotide sequence of interest, and comparing the first and second expression levels of the nucleotide sequences of interest.
  • the invention provides a method of identifying an arthritis modulating compound.
  • the method involves the steps of obtaining a first synovial fluid monocyte sample from the subject, assaying a first expression level of a nucleotide sequence of interest in the sample, administering a compound of interest, obtaining a second synovial fluid monocyte sample from the subject, assaying a second expression level of the nucleotide sequence of interest, and comparing the first and second expression levels of the nucleotide sequences of interest.
  • Figure 1 presents expression profiles for the indicated chemokines in peripheral blood monocytes (PBMC, panels A and B) and synovial fluid monocytes (SFMC, panels C and D) obtained from juvenile arthritis patients. Expression of the indicated nucleotide sequences of interest in the tissue samples is shown relative to expression of the nucleotide sequence of interest in peripheral blood monocytes obtained from healthy individuals. Samples were obtained from patients with polyarticular juvenile arthritis (poly, hatched bars), pauciarticular juvenile arthritis (pauci, white bars), and juvenile spondyloarthropathy (JSpA, solid bars).
  • FIG. 1 presents relative expression values of various nucleotide sequences of interest in patients exhibiting a pauciarticular course, a polyarticular course, and in juvenile spondyloarthropathy (JSpA).
  • JSpA juvenile spondyloarthropathy
  • the present invention provides methods of classifying disease, particularly juvenile arthritides, determining disease progression, predicting disease course, and identifying anti-arthritic compounds.
  • Compositions of the invention include kits for performing the methods of the invention and anti-arthritic compounds identified by a method of the invention.
  • the invention relates to differential expression of nucleotide sequences of interest in peripheral blood monocytes (PBMCs) and synovial fluid monocytes (SFMCs) in the various types of juvenile arthritis.
  • PBMCs peripheral blood monocytes
  • SFMCs synovial fluid monocytes
  • Juvenile arthritis classification systems include, but are not limited to, criteria developed by the ACR, EULAR, and the International League of Associations for Rheumatology. Classification criteria among these three systems vary, but in all three the subtypes of juvenile arthritis are generally characterized by the number of affected joints within six months of onset. Juvenile arthritis classifications include, but are not limited to, pauciarticular arthritis, polyarticular arthritis, systemic onset juvenile rheumatoid arthritis, and juvenile onset spondyloarthropathy.
  • Subject is intended a mammal, e.g., a human, or an experimental or animal or disease model or mammalian tissue or mammalian cells. Suitable subjects include mammals, particularly humans, exhibiting a juvenile arthritis, tissue obtained from a mammal exhibiting a juvenile arthritis, cells obtained from a mammal exhibiting a juvenile arthritis, and cells cultured from a mammal exhibiting a juvenile arthritis.
  • the subject can also be a non-human animal such as, but not limited to, a horse, hamster, guinea pig, mouse, rabbit, dog, pig, goat, cow, rat, monkey, chimpanzee, sheep, or other domestic animal.
  • biological sample is intended a sample collected from a subject including, but not limited to, tissues, cells, mucosa, fluid, scrapings, hairs, cell lysates, and secretions, particularly peripheral blood monocytes and synovial fluid monocytes.
  • a peripheral blood monocyte sample comprises at least one monocyte cell obtained from peripheral blood.
  • a synovial fluid monocyte sample comprises at least one monocyte obtained from synovial fluid.
  • Biological samples such as peripheral blood monocyte samples and synovial fluid monocyte samples can be obtained by any method known to one skilled in the art. Further, biological samples such as, but not limited to peripheral blood monocytes and synovial fluid monocyte samples can be enriched, purified, or isolated by any method known to one skilled in the art. [0033] An "isolated” or “purified” biological sample, particularly a peripheral blood monocyte sample or synovial fluid monocyte sample is substantially or essentially free from components that normally accompany or interact with the sample as found in its naturally occurring environment. Thus, an isolated or purified PBMC or SFMC sample is substantially free of other cell types.
  • a PBMC or SFMC sample that is substantially free of extraneous material includes preparations of monocytes having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating cells.
  • Methods of isolating peripheral blood monocytes from whole blood include, but are not limited to, Ficoll gradient centrifugation and ultracentrifugation (de Jager et al. (2003) Clin. & Diag. Lab. Immun. 10:133-139, herein inco ⁇ orated by reference in its entirety).
  • Methods of isolating synovial fluid mononuclear cells are known in the art and include, but are not limited to, Ficoll gradient centrifugation of synovial fluid.
  • Methods of isolating synovial fluid mononuclear cells include, but are not limited to, Ficoll gradient centrifugation of synovial fluid.
  • the process of isolating monocytes from a source sample begins within about 24 hours of collection, preferably within about 12 hours of collection, more preferably within about 8 hours of collection, yet more preferably within about 4 hours of collection of the source sample.
  • Methods of assaying expression levels include, but are not limited to, qualitative Western blot analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, RT-PCR, 2-D gel electrophoresis, microarray analysis, in situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex RT-PCR, immunohistochemical assays, radioimmunoassay, colorimetric analysis, immunoradiometric assays, positron emission tomography, Northern blotting, fluorometric assays, fluorescence activated cell sorter staining of permeabilized cells, radioimmunosorbent assays, real-time PCR, hybridization assays, sandwich immunoassays, flow cytometry, SAGE, differential amplification, or electronic analysis.
  • Nucleotide sequences of interest in the present invention include numerous inflammatory related compounds. Nucleotide sequences of interest in the present invention include, but are not limited to, CXCL1 (SEQ ID NO:l), CXCL2 (SEQ ID NO:3), CXCL3 (SEQ ID NO:5), CXCL4 (SEQ ID NO:7), CXCL5 (SEQ ID NO:9), CXCL7 (SEQ ID NO:ll), CXCL8 (SEQ ID NO:13), CXCL9 (SEQ ID NO:15), CXCLIO (SEQ ID NO:17), CXCL11 (SEQ ID NO:19), CXCL13 (SEQ ID NO:21), VEGF (SEQ ID NO:55), CCL2 (SEQ ID NO:25), AREG (SEQ ID NO:35), PBEF (SEQ ID NO:23), PHLDA (SEQ ID NO:27), SERPINB2 (SEQ ID NO:29), TGIF (SEQ ID NO:31
  • cytokines include, but are not limited to, cytokines and chemokines.
  • a cytokine is a general term for a mediator released primarily but not exclusively by a cell population of the immune system as a response to a specific stimulating agent, e.g., a specific antigen or an alloantigen; or a non-specific, polyclonal activator, e.g. an endotoxin or other cell wall components.
  • a specific stimulating agent e.g., a specific antigen or an alloantigen
  • a non-specific, polyclonal activator e.g. an endotoxin or other cell wall components.
  • Chemokines are members of the large superfamily of inducible small cytokines and can be divided into at least four groups according to a conserved structural motif of the first two closely paired cysteines within their amino acid sequence.
  • the CXC chemokines have a single amino acid separating the first two cysteines.
  • CXCL chemokines represent specific ligands of the CXCRs (Cys-X-Cys receptor).
  • CXCL chemokines including, but not limited to, CXCL1 (SEQ ID NO:l), CXCL2 (SEQ ID NO:3), CXCL3 (SEQ ID NO:5), CXCL4 (SEQ ID NO:7), CXCL5 (SEQ ID NO:9), CXCL7 (SEQ ID NO:l 1), CXCL8 (SEQ ID NO:13), CXCL9 (SEQ ID NO:15), CXCLIO (SEQ ID NO:17), CXCL11 (SEQ ID NO:19), and CXCL13 (SEQ ID NO:21), and VEGF (Genbank NM003376; SEQ ID NO:55).
  • a nucleotide sequence of interest is any nucleotide sequence having a nucleotide sequence set forth in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55; a nucleotide sequence having at least 90% identity to a nucleotide sequence set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55; a nucleotide sequence having an amino acid sequence set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 56; a nucleotide sequence having an amino acid sequence having at least 90% identity
  • fragments and variants of the nucleotide sequences of interest and proteins encoded thereby are also encompassed by the present invention.
  • fragment is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby.
  • Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence exhibit activity Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
  • fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.
  • a fragment of a nucleotide sequence of interest that encodes a biologically active portion of a nucleotide sequence of interest will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 415, or up to the total number of amino acids present in a full-length nucleotide sequence of interest.
  • Fragments of a nucleotide sequence of interest that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a polypeptide encoded by a nucleotide sequence of interest.
  • a fragment of a nucleotide sequence of interest may encode a biologically active portion of a nucleotide sequence of interest or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • a biologically active portion of a nucleotide sequence of interest can be prepared by isolating a portion of one of the nucleotide sequences of the invention, expressing the encoded portion of the protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the protein.
  • Nucleic acid molecules that are fragments of a nucleotide sequence of interest comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850,3900, 4000, 4050, 4100, 4150, 4200, 4212 nucleotides, or up to the number of nucleot
  • Fragments of interest include, but are not limited to, the nucleotide sequences set forth in SEQ ID NOS:37-54.
  • variants are intended substantially similar sequences.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the nucleotide sequence of interest polypeptides.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a nucleotide sequence of interest.
  • variants of a particular nucleotide sequence of the invention will have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
  • variant protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymo ⁇ hism or from human manipulation.
  • Biologically active variants of a native polypeptide encoded by a nucleotide sequence of interest will have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions.
  • amino acid sequence variants of the polypeptides encoded by the nucleotide sequences of interest can be prepared by mutations in the DNA.
  • Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl Acad. Sci. USA 52:488-492; Kunkel et al. (1987) Methods in Enzymol. 54:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired expression pattern.
  • nucleotide sequences disclosed herein can be used to identify corresponding sequences in cells, tissues, and animals. In this manner, methods such as PCR, hybridization, microarrays, and the like can be used to assay expression of such sequences based on their sequence homology to the sequences set forth herein. These techniques may be used as a diagnostic assay to determine expression levels of the nucleotide sequences of interest in an animal or animal cell.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any animal of interest.
  • Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al, eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds.
  • PCR PCR Strategies
  • nested primers single specific primers
  • degenerate primers gene-specific primers
  • vector-specific primers partially-mismatched primers
  • hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the nucleotide sequences of interest.
  • nucleotide sequence of interest may be used as a probe capable of specifically hybridizing to corresponding nucleotide sequences of interest.
  • probes include sequences that are unique among nucleotide sequences of interest and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
  • Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances.
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5 X to 1 X SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. [0051] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA--DNA hybrids, the T m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem.
  • T m 81.5°C+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
  • T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • sequences that have inflammatory related activity and which hybridize under stringent conditions to the nucleotide sequences of interest disclosed herein, or to fragments thereof, are encompassed by the present invention.
  • sequences will be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% to 99% homologous or more with the disclosed sequences. That is, the sequence identity of sequences may range, sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Sequence comparison programs include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins et al (1988) Gene 73:237-244 (1988); Higgins et al (1989) CABIOS 5:151-153; Co ⁇ et et a/. (1988) Nucleic Acids Res.
  • ALIGN program is based on the algorithm of Myers and Miller (1988) supra.
  • a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.
  • the BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra.
  • Gapped BLAST in BLAST 2.0
  • PSI- BLAST in BLAST 2.0
  • PSI- BLAST in BLAST 2.0
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence similarity or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • nucleotide sequences are substantially identical.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • stringent conditions encompass temperatures in the range of about 1°C to about 20°C lower than the T m , depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453.
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • Expression of the nucleotide sequences of interest in the present invention differs between cell types and among classifications of juvenile arthritis. Expression differences include, but are not limited to, the following variations in expression. Expression of CXCL1, CXCL2, CXCL3, and CXCL8 in peripheral blood monocytes (PBMCs) from polyarticular subjects was higher than in PBMCs from pauciarticular subjects and healthy controls. However, expression levels of these chemokines were equivalent in synovial fluid monocyte (SFMCs) samples from patients with the various disease classifications.
  • PBMCs peripheral blood monocytes
  • SFMCs synovial fluid monocyte
  • CXCL9, CXCLIO, and CXCL11 were lower in SFMCs from polyarticular patients compared with SFMCs from pauciarticular patients.
  • Expression of CXCL4 and CXCLIO in SFMCs from juvenile onset spondyloarthropathies was lower than expression in SFMCs from pauciarticular patients.
  • Expression of the angiostatic chemokines in PBMCs was essentially the same between juvenile arthritis classifications.
  • the methods of the invention are based on the differential expression of one or more nucleotide sequences of interest in one or more cell types.
  • "Differential expression" as used herein refers to both quantitative as well as qualitative differences in the genes' temporal and or tissue expression patterns.
  • a differentially expressed nucleotide sequence of interest may have its expression activated or completely inactivated among the disease classifications or under control versus experimental conditions.
  • a qualitatively regulated gene will exhibit an expression pattern within a given tissue or cell type which is detectable in either healthy subjects or subjects with a juvenile arthritides.
  • the expression of a nucleotide sequence of interest is detectable in 0, 1, 2, 3, 4 or more classifications of juvenile arthritis.
  • such a qualitatively regulated gene will exhibit an expression pattern within a given tissue or cell type which is detectable in either control or experimental subjects, but is not detectable in both.
  • a differentially expressed gene may have its expression modulated, i.e., quantitatively increased or decreased, among the juvenile arthritis subtypes, in normal versus disease states, or under control versus experimental conditions.
  • Transcript levels of differentially expressed genes may vary by 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more.
  • expression pattern is intended a description of the relative expression levels of one or more nucleotide sequences in one or more cell types.
  • a standard expression pattern is a predetermined description of the relative expression levels of one or more nucleotide sequences in one or more cell types.
  • a standard expression pattern might describe the relative expression levels of one or more nucleotide sequences of interest in peripheral blood monocytes, in synovial fluid monocytes, or in both peripheral blood monocytes and synovial fluid monocytes.
  • Expression patterns of the nucleotide sequences of interest in PBMCs and SFMCs differ among the various classifications of juvenile arthritis.
  • the expression patterns indicated in Figure 1 and Figure 2 are examples of standard expression patterns suitable for determining the classification of juvenile arthritis that a patient exhibits. Expression levels of one or more nucleotide sequences of interest in samples obtained from a subject are compared to standard expression patterns indicative of juvenile arthritis classification.
  • Disease progression or “disease course” is intended the physiological events related to a disease or disorder that occur during the period after the initial presentation in a subject or patient exhibiting a disease or disorder. With respect to juvenile arthritis, the number of affected joints beyond the first six months of disease is used to describe the disease course or progression.
  • a pauciarticular course involves four or fewer joints; a polyarticular course involves five or more joints.
  • Systemic onset juvenile rheumatoid arthritis often presents pauciarticularly but typically progresses polyarticularly.
  • Juvenile onset spondyloarthropathy JSpA
  • Identifying the disease course or disease progression that an untreated patient would experience allows practitioners to identity those patients who would benefit most from aggressive early intervention. Identifying compounds or agents that alter disease progression is an embodiment of the invention.
  • Expression profiles of the nucleotide sequences of interest may be used to analyze disease progression or disease course in a subject with juvenile arthritis.
  • Juvenile arthritis phenotypes include, but are not limited to, expression of the nucleotide sequences of interest, number of affected joints, apoptotic tendency, erythrocyte sedimentation rate, C reactive protein levels, IL15 expression levels, joint pain, joint swelling, joint stiffness, irritability, ulceris, iridocyclitis, uveitis, fevers, rashes, rheumatoid factor presence, synovial thickening, synovial joint space expansion, effusion in the suprapatellar pouch, bone mineral content, bone mineral density, (See Petty, R.E. and Cassidy, J.T. (2001) The Juvenile Idiopathic Arthritides.
  • Methods of measuring juvenile arthritis phenotypes include, but are not limited to, methods of assaying expression described elsewhere herein, TUNEL, arthrosonography, and dual energy x-ray abso ⁇ tiometry. See Bendtzen et al. (2003) Clin. Exp. Immunol. 134:151-158; Smolewska et al. 20Q ⁇ ) Ann. Rheum. Dis. 62:761-763; Lien et al. (2003) Arthritis Rheum. 48:2214-2223, the Childhood Health Assessment Questionnaire, Textbook ofPediatric Rheumatology. 4 th ed. J. T. Cassidy and R.E. Petty, ed.
  • nucleotide sequence of interest expression modulating compound is a compound that modulates expression of a nucleotide sequence of interest.
  • Modulation maybe an increase or decrease in expression of the nucleotide sequence of interest in one or more samples from a subject.
  • a nucleotide sequence of interest expression modulating compound will modulate expression of a nucleotide sequence of interest by at least 1%, 5%, preferably 10%, 20%, more preferably 30%, 40%, 50%, 60%, yet more preferably 70%, 80%, 90%, or 100% as compared to an untreated or placebo treatment effect. Modulation of expression of a nucleotide sequence of interest may occur in only one tissue or it may occur in multiple tissues. Methods for assaying expression of nucleotide sequences of interest are described elsewhere herein.
  • a first biological sample is obtained from a subject, particularly a subject exhibiting juvenile arthritis.
  • a first biological sample is a first peripheral blood monocyte sample, a first synovial fluid monocyte sample, or both a first peripheral blood monocyte sample and a first synovial fluid monocyte sample.
  • a compound of interest is administered to the subject. After administration of either the compound of interest or a placebo, the subject is incubated for a period of time. The period of time will have a predetermined duration appropriate to analysis of a juvenile arthritis phenotype.
  • a second biological sample is obtained from the subject.
  • a second biological sample is a second peripheral blood monocyte sample, a second synovial fluid monocyte sample, or both a second peripheral blood monocyte sample and a second synovial fluid monocyte sample.
  • Monitoring of expression of a nucleotide sequence of interest may occur continuously; at a single interval; or at multiple intervals, such as, but not limited to, hourly, daily, weekly, and monthly.
  • any method of assaying expression of a nucleotide sequence of interest known in the art may be used to monitor the effects of the compound of interest on a transgenic animal of the invention.
  • the nucleotide sequences of interest in the invention may be used to identify an arthritis modulating compound.
  • An "arthritis modulating compound” is a compound that modulates an arthritic phenotype. Modulation may be an increase or decrease in an arthritic phenotype.
  • An arthritis modulating compound will modulate a arthritic phenotype by at least 1%, 5%, preferably 10%, 20%, more preferably 30%, 40%, 50%, 60%, yet more preferably 70%, 80%, 90%, or 100% as compared to an untreated or placebo treatment effect.
  • a first peripheral blood monocyte sample and a first synovial fluid monocyte sample are obtained from a subject exhibiting juvenile arthritis or cells or tissues from a mammal exhibiting juvenile arthritis.
  • a compound of interest is administered to the subject. After administration of either the compound of interest or a placebo, the subject is incubated for a period of time. The period of time will have a predetermined duration appropriate to analysis of the phenotype.
  • Such durations include, but are not limited to, 30 seconds; 1, 5, 10, 30, or 60 minutes; 8, 12, 24, 36, or 48 hours; 3, 4, 5, 6, or 7 days; 2, 3, or 4 weeks; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months; up to 3 years.
  • a second peripheral blood monocyte sample and/or a second synovial fluid monocyte sample is obtained.
  • Monitoring of an arthritis phenotype may occur continuously; at a single interval; or at multiple intervals, such as, but not limited to, hourly, daily, weekly, and monthly. Any method of assaying an arthritis phenotype known in the art may be used to monitor the effects of the compound of interest on the subject.
  • administer is used in its broadest sense and includes any method of introducing a compound into a subject such as, but not limited to, a human, mouse, rabbit, dog, pig, goat, cow, rat, monkey, chimpanzee, or sheep. This includes producing polypeptides or polynucleotides in vivo as by transcription or translation in vivo of polynucleotides that have been exogenously introduced into a subject.
  • a “compound” comprises, but is not limited to, nucleic acid molecules, aldosterone antagonists, polypeptides, peptides, peptidomimetics, glycoproteins, transcription factors, small molecules, chemokine receptors, antisense nucleotide sequences, chemokine receptor ligands, lipids, antibodies, receptor inhibitors, ligands, sterols, steroids, hormones, chemokine receptor agonists, chemokine receptor antagonists, agonists, antagonists, ion-channel modulators, diuretics, enzymes, enzyme inhibitors, carbohydrates, deaminases, deaminase inhibitors, G-proteins, G-protein receptor inhibitors, ACE inhibitors, hormone receptor modulators, alcohols, reverse transcriptase inhibitors, neurotransmitter inhibitors, neurotransmitter receptor modulators, hormones
  • a compound may additionally comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycer
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a carboxypeptidase protein or anti- carboxypeptidase antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a carboxypeptidase protein or anti- carboxypeptidase antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods.
  • the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • compositions for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. [0083] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Arthritis modulating compounds identified by the methods of this invention may be used in the treatment of human individuals.
  • the invention provides kits for performing the methods of the invention. Such kits comprise a collection reagent and a detection reagent.
  • Collection reagent is intended any substance that facilitates collection of the indicated substance. Collection reagents that facilitate the reaction may or may not participate in the purification or enrichment of the desired cell type in the sample. Collection reagents include, but are not limited to, syringes, needles, tubing, butterfly syringes, plastic vials, glass vials, centrifuges, Ficoll, ultracentrifuges, vessels, such as microfuge tubes and multiwell plates; measuring devices, such as micropipette tips and capillary tubes; filters; separation devices such as microfuge tube filter inserts, vacuum apparati, purification resins, magnetic beads, and columns; reagents; compounds; solutions; molecules; buffers; inhibitors; chelating agents; ions; terminators; stabilizers; precipitants; solubilizers; acids; bases; salts; reducing agents; oxidizing agents; enzymes; catalysts; and denaturants.
  • detection reagent any substance that facilitates detection of the expression of a nucleotide sequence of interest.
  • Detection reagents include, but are not limited to, microchips, microarrays, primers, probes, antibodies, nucleic acid probes, fluorescently labeled primers, fluorescently labeled antibodies, radiolabeled antibodies, radiolabeled primers, radiolabeled probes, expression analyzing reagents, and other reagents known to one of skill in the art.
  • a concentrated detection reagent is provided in kits of the invention.
  • the concentration of a detection reagent provided in a kit of the invention may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more fold concentrated than the desired concentration of the detection reagent in a detection reaction.
  • a kit provides a detection reagent and a transfer component.
  • a "transfer component” is a material that facilitates transfer of the detection reagent to a processing facility. Transfer components include but are not limited to, packages, boxes, shipping labels, and envelopes. Use of a processing facility to process the detection reagent enhances instrumentation and protocol standardization which may be of particular benefit when the detection reagent is a microchip or microarray.
  • Example 2 Expression Profiling of Peripheral Blood Monocyte Samples and Synovial Fluid Monocytes
  • Biotinylated cRNA was synthesized from total RNA using manufacturer's recommended protocols (Enzo; Farmingdale NY). The labeled cRNA was processed as recommended by the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix; Santa Clara, CA). Labeled cRNA was hybridized to Affymetrix U95A chips. Quality was assessed arid expression values were derived using Microarray Suite 5.0 (MAS 5.0; Affymetrix).
  • Real-time RT-PCR was performed on total RNA prepared separately from that used in the microarray expression analysis. The real-time RT-PCR was performed using Assays-on-Demand reagents from Applied Biosystems, Foster City CA and using tl e manufacturer's recommended protocols. Real time RT-PCR was used to amplify CXCL3 (Hs00171061_ml), CXCL8 (Hs00174103_ml), and GAPDH (Hs00174103_ml) from total RNA.

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Abstract

La présente invention porte sur un procédé visant à déterminer la classification d'une maladie, en particulier l'arthrite juvénile. L'invention porte également sur une méthode d'analyse de l'évolution de la maladie chez un sujet souffrant de l'arthrite juvénile. L'invention porte aussi sur des modèles d'expression de certaines séquences nucléotidiques liées à une inflammation qui diffèrent parmi les diverses classifications de l'arthrite juvénile telles que, mais exclusivement, l'arthrite pauciarticulaire, l'arthrite polyarticulaire, l'apparition de la spondyloarthrophathie juvénile, et l'apparition de l'arthrite rhumatoïde juvénile systémique.
PCT/US2004/035255 2003-10-23 2004-10-22 Procedes de determination de la classification de l'arthrite juvenile Ceased WO2005056753A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1941057A4 (fr) * 2005-10-07 2009-11-11 Baylor Res Inst Diagnostic d'une arthrite juvenile idiopathique avec debut systemique par analyse de puces a adn a base de leucocytes sanguins

Non-Patent Citations (3)

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Title
BARNES ET AL: 'Angiogenic/Angiostatic Chemokine Gene Expression in Juvenile Arthritis' OASIS September 2003, *
GATTORNO ET AL: 'Synovial fluid T cell clones from oligoarticular juvenile arthritis patients display a prevalent Th1/Th0-type pattern of cytokine secretion irrespective of immunophenotype' CLIN. EXP. IMMUNOL. vol. 109, 1997, pages 4 - 11 *
RUSCHPLER ET AL: 'High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis' ARTHRITIS RES. THER. vol. 5, 26 June 2003, pages R241 - R252 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1941057A4 (fr) * 2005-10-07 2009-11-11 Baylor Res Inst Diagnostic d'une arthrite juvenile idiopathique avec debut systemique par analyse de puces a adn a base de leucocytes sanguins

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