# Patent

## Title
Methods of diagnosing and treating rheumatoid arthritis and osteoarthritis

## Abstract

The invention provides a novel cell that is a precursor of a fibroblast-like synovial cell. The novel cell is a circulating synovial tissue cell that stains positive for collagen, CD34, CD45, prolyl 4-hydroxylase and CD14. The invention also relates to methods of diagnosing or monitoring rheumatoid arthritis and/or osteoarthritis using gene expression profiles, protein expression profiles, and/or protein phosphorylation profiles of different cell types, including the novel precursor, CD3+ cells, synovial tissue fibroblast-like synovial cells and fibrocytes. The invention also includes methods to identify substances to treat or prevent rheumatoid arthritis and/or osteoarthritis.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 USC 119(e) from U.S. provisional application No. 60/808,294, filed on May 25, 2006, which is incorporated by reference herein its entirety.


FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under contract HV028183 awarded by the National Institutes of Health. The Government has certain rights in this invention.


FIELD OF THE INVENTION
The invention relates to a novel cell that is a precursor of a fibroblast-like synovial cell. The invention also relates to methods of diagnosing or monitoring rheumatoid arthritis and/or osteoarthritis using gene expression profiles, protein expression profiles, and/or protein phosphorylation profiles of different cell types, including the novel precursor, CD3+ cells, fibroblast-like synovial cells, and fibrocytes.
BACKGROUND OF THE INVENTION
Rheumatoid arthritis (RA) is a common, relapsing autoimmune disease affecting 0.8-1% of the population worldwide (1) (2). RA presents clinically with joint swelling, deformity, pain, stiffness, and weakness (3). The primary sites of tissue damage are joints, but systemic involvement of the eyes, kidneys, chest and lungs may also occur (4). The rheumatoid synovial environment is an area of intense immunological activity. The cellular composition of the affected RA joint is characterized by proliferation of synovial lining cells, pannus accumulation over articular cartilage and the infiltration of inflammatory cells, including mononuclear cells and lymphocytes. Fibroblast-like synovial (FLS) cells are thought to be responsible for pannus formation and contribute to bone and cartilage destruction.
One of the hallmarks of RA is synovial hyperplasia. Two critical resident cells types in affected synovial tissue (ST) are: a CD68+/MHCII+ macrophage-like synoviocyte (MLS) and a CD68−/MHCII-FLS cell (5). The intimal layer increases from several cells to 15 cells deep, due to increased FLS cell numbers, through a combination of increased proliferation, decreased apoptosis and decreased senescence (5). FLS cells synthesize and secrete many pro-inflammatory mediators—cytokines, chemokines, growth factors—that are involved in autocrine and paracrine regulation of inflammation (5) and, therefore, are critical effectors in regulating the inflammatory response in RA. FLS cells are found in the intima and subintima, and FLS cells in RA are thought to transform into cells that proliferate in an anchorage-independent manner, lack contact inhibition and secrete cytokines constitutively. Many growth factors, such as PDGF, bFGF, TGF-β and activin are expressed in RA and drive fibroblast proliferation in vitro (6) (7) (8) (9) (10).
SUMMARY OF THE INVENTION
The present inventors have identified a precursor of a fibroblast-like synovial cell that comprises a circulating cell that stains positive for collagen, CD34, CD45, prolyl 4-hydroxylase and CD14. The activation status of the novel precursor is useful to diagnose or monitor rheumatoid arthritis in a subject.
Accordingly, the invention includes an isolated precursor of a fibroblast-like synovial cell, comprising a circulating cell that stains positive for collagen, CD34, CD45, prolyl 4-hydroxylase and CD14.
The invention also includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the number of isolated precursor cells of the invention in a sample from the subject; and
        (b) comparing the number of isolated precursor cells from the sample with a control;
    
    


wherein a difference in the number of isolated precursor cells in the sample from the subject as compared to the control is indicative of rheumatoid arthritis.
Another aspect of the invention, is a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the activation state of the isolated precursor cell of the invention from a sample from the subject; and
        (b) (b) comparing the activation state of the isolated precursor cell from the sample with a control;
    
    


wherein the activation state of the precursor cell is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the isolated precursor cell as compared to the control is indicative of rheumatoid arthritis.
The invention also includes the use of the isolated precursor cell of the invention to diagnose or monitor rheumatoid arthritis.
An additional aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the number of isolated precursor cells of the invention in a sample from a subject treated with a substance; and
        (b) comparing the number of isolated precursor cells from the sample with a control;
    
    


wherein a difference in the number of isolated precursor cells in the sample from the subject as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
A further aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the activation state of the isolated precursor cell of the invention from a sample from a subject treated with a substance; and
        (b) comparing the activation state of the isolated precursor cell from the sample with a control;
    
    


wherein the activation state of the precursor cell is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the isolated precursor cell as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
In addition, the invention includes the use of the isolated precursor cell of the invention to identify a substance to treat or prevent rheumatoid arthritis.
The inventors have also analyzed the activation status of circulating CD3+ cells and have determined that increases in phosphorylation of various signaling molecules correlates with the progression of rheumatoid arthritis.
Accordingly, the invention includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the activation state of a CD3+ cell from a sample from the subject; and
        (b) comparing the activation state of the CD3+ cell from the sample with a control;
    
    


wherein, the activation state of the CD3+ cell is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the CD3+ cell as compared to the control is indicative of rheumatoid arthritis.
The invention also includes the use of a CD3+ cell to diagnose or monitor rheumatoid arthritis in subject.
Another aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis comprising the steps:

    
    
        (a) determining the activation state of a CD3+ cell from a sample from a subject treated with a substance; and
        (b) comparing the activation state of the CD3+ cell from the sample with a control;
    
    


wherein the activation state of the CD3+ cell is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the CD3+ cell as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
The invention also includes the use of a CD3+ cell to identify a substance to treat or prevent rheumatoid arthritis.
The inventors have also characterized the gene and protein expression profiles of synovial tissue fibroblast-like synovial cells, and the protein phosphorylation profiles of these cells in samples from individuals with rheumatoid arthritis or osteoarthritis.
Accordingly, the invention includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the gene expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the gene expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the gene expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of rheumatoid arthritis.
Another aspect of the invention is a method of diagnosing or monitoring osteoarthritis in a subject, comprising the steps:

    
    
        (a) determining the gene expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the gene expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the gene expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of osteoarthritis.
An additional aspect of the invention is a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the protein expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the protein expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of rheumatoid arthritis.
A further aspect of the invention is a method of diagnosing or monitoring osteoarthritis in a subject, comprising the steps:

    
    
        (a) determining the protein expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the protein expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of osteoarthritis.
An additional aspect of the invention is a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the protein phosphorylation profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of rheumatoid arthritis.
Another aspect of the invention is a method of diagnosing or monitoring osteoarthritis in a subject, comprising the steps:

    
    
        (a) determining the protein phosphorylation profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of osteoarthritis.
The methods of the invention can also be used to identify substances to treat or prevent rheumatoid arthritis or osteoarthritis.
A further aspect of the invention is a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the number of circulating fibrocytes in a sample from the subject; and
        (b) comparing the number of fibrocytes from the sample with a control;
    
    


wherein a difference in the number of fibrocytes in the sample from the subject as compared to the control is indicative of rheumatoid arthritis.
Another aspect of the invention, is a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the activation state of a circulating fibrocyte from a sample from the subject; and
        (b) comparing the activation state of the fibrocyte from the sample with a control;
    
    


wherein the activation state of the fibrocyte is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the fibrocyte as compared to the control is indicative of rheumatoid arthritis.
The invention also includes using circulating fibrocytes to diagnose or monitor rheumatoid arthritis.
An additional aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the number of circulating fibrocytes in a sample from a subject treated with a substance; and
        (b) comparing the number of fibrocytes from the sample with a control;
    
    


wherein a difference in the number of fibrocytes in the sample from the subject as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
A further aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the activation state of circulating fibrocytes from a sample from a subject treated with a substance; and
        (b) comparing the activation state of the fibrocyte from the sample with a control;
    
    


wherein the activation state of the fibrocyte is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the fibrocyte as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
In addition, the invention includes the use of circulating fibrocytes to identify a substance to treat or prevent rheumatoid arthritis.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The invention will now be described in relation to the drawings in which:
FIG. 1 is heat map representation showing the gene expression levels of synovial tissue fibroblast-like synovial cells in controls or subjects with rheumatoid arthritis or osteoarthritis. Red indicates increased expression levels above the mean and increasing blue intensity indicates reduced expression. The intensity of the red or blue corresponds to the extent of the change from normal.
FIG. 2 is a heat map representation showing the phospho-protein signature profiles of synovial tissue fibroblast-like synovial cells (ST FLS) from subjects with rheumatoid arthritis or osteoarthritis. Cell lysates derived from ST FLS cells from affected joints from 2 OA patients (LHS panel) and 3 RA patients (RHS panel) were analyzed using customized BD phospho-protein PowerBlots. Heat map representation: phospho-protein expression profiles are represented as a “heat-map” in which red indicates increased expression levels above the mean and increasing blue intensity indicates reduced expression. The intensity of the red or blue corresponds to the extent of the change from normal. Each array map represents 80 phosphospecificities. Similar patterns are indicative of similar phospho-protein profiles. This is evident for the 2 representative OA ST FLS cell specimens. These are distinguishable from the 3 RA ST FLS cell signature profiles. Interestingly, in regard to the RA specimens, although exhibiting similar phosphorylation-activation of many signaling effectors and kinases, there are clusters of distinctive patterns, allowing for stratification of the RA specimens into 3 sub-groups, exemplified by the 3 profiles provided.
FIG. 3 shows the protein expression in synovial tissue fibroblast-like synovial cells derived from individuals with rheumatoid arthritis or osteoarthritis.
FIG. 4 shows the immunostaining results of peripheral blood fibrocytes for various markers. (A) FACS analysis of a population of peripheral blood fibrotcyes. (B) Confocal microscopy of a single peripheral blood fibrocyte. Images were collected using an upright Leica SP2 confocal laser-scanning microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany), 100× oil immersion lens (1.4 NA) and 4× digital zoom.
FIG. 5 shows the results of an intracellular analysis of phospho-STAT3 in CD3−, collagen+ peripheral blood fibrocytes.
FIG. 6 shows the results of an intracellular analysis of phospho-STAT5 in CD3−, collagen+ peripheral blood fibrocytes.
FIG. 7 shows the results of an intracellular analysis of phospho-ERK in CD3−, collagen+ peripheral blood fibrocytes.
FIG. 8 shows the results of an intracellular analysis of phospho-p38 MAPK in CD3−, collagen+ peripheral blood fibrocytes.
FIG. 9 shows protein phosphorylation profiles of sub-populations and primary cells.
FIG. 10 shows the results of an intracellular analysis of phospho-STAT3 and phospho-p38 in CD3+ cells from healthy individuals, early rheumatoid arthritis patients and late rheumatoid arthritis patients.
FIG. 11 depicts a signalizing cascade.
FIG. 12 shows circulating fibrocytes in CIA. Mice with CIA exhibit obvious swelling of the paws/joints compared to naïve animals (A-1, II). Affected joints were scored on a scale of 0-16 and the cumulative disease score is shown in panel B. Mice with CIA cumulative score of 6/individual paw score of 2, show obvious swelling and cellular infiltrates in the dermis (C III, magnification 100×; C IV, magnification 200×*) compared to control animals (C I, magnification 100×; C II, magnification 200×). The majority of the inflammatory infiltrate were neutrophils (PMN) but macrophages (M), lymphocytes (L) and plasma cells (P) were also observed (C V, 1000× magnification). Swelling (**) and early inflammatory infiltrates (&) were observed in the intra-articular spaces (C VI, 200× magnification). PBMC were collected by cardiac puncture at different stages of disease and FACS analysis performed to detect the α-SMA/CI fibrocyte population (D). Panel E describes the α-SMA/CI fibrocyte population on day 30. Large granular cells were gated by FSC/SSC and double positive cells are shown. Notably, the number of circulating fibrocytes was higher in mice with higher disease scores.
FIG. 13 shows evidence of increased p-STAT5 in circulating fibrocytes from animals with early stages of CIA. PBMC were isolated from the peripheral blood of control and CIA mice at stage 1-2. The cells were stained for Collagen I-Alexa647, α-SMA-FITC and p-STAT5-PE and analyzed by FACS. The ColI+ cells were gated and the percentage of α-SMA/p-STAT5 double positive cells are shown (A, R2 gate and B).
FIG. 14 shows immunohistochemistry of paraffin embedded joints from mice with collagen induced arthritis stained with α-SMA-FITC (green) and CD45-PE (red). α-SMA also stains smooth muscle and is clearly identified surrounding an artery (open arrow). A profound cellular influx of leukocytes is evident (*) in the CIA joint tissue and the majority of these cells do not stain with o-SMA (overlay). α-SMA/CD45 fibrocytes/myofibroblasts were easily identified in the CIA joints (closed arrow) and were more abundant than in normal joints.
FIG. 15 shows immunohistochemistry of plastic embedded joints from mice with collagen induced arthritis stained with α-SMA-FITC (green) and CD45-PE (red). α-SMA/CD45 double positive fibrocytes/myofibroblasts were identified in the CIA joints.



DETAILED DESCRIPTION OF THE INVENTION
The inventors have discovered a precursor of a fibroblast-like synovial cell. This cell is a circulating cell and can migrate to affected joints in subjects with rheumatoid arthritis, and is able to transform into resident myofibroblasts and fibroblast-like synovial cells. The precursor cell stains positive for collagen, CD34, CD45, prolyl 4-hydroxylase and CD14, and can be isolated from the circulatory system, for example from peripheral blood mononuclear cells from healthy individuals and individuals with rheumatoid arthritis.
Accordingly, the invention provides an isolated precursor of a fibroblast-like synovial cell, comprising a circulating cell that stains positive for collagen, CD34, CD45, prolyl 4-hydroxylase and CD14.
The term “isolated precursor of a fibroblast-like synovial cell” as used herein refers to the precursor cells of the invention substantially free of other cell types. In another embodiment, the cells are also substantially free of cellular debris or other cellular material, or culture medium.
The invention includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the number of isolated precursor cells of the invention in a sample from the subject; and
        (b) comparing the number of isolated precursor cells from the sample with a control;
    
    


wherein a difference in the number of isolated precursor cells in the sample from the subject as compared to the control is indicative of rheumatoid arthritis.
The inventors also analyzed the activation state of this circulating precursor cell in healthy individuals and subjects with early rheumatoid arthritis and subjects with later stages of disease. The activation state of the precursor cell was determined by analyzing the phosphorylation levels of signaling molecules, including STAT3, STAT5, ERK and/or p38 MAPK.
Accordingly, the invention includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the activation state of the isolated precursor cell of the invention from a sample from the subject; and
        (b) comparing the activation state of the isolated precursor cell from the sample with a control;
    
    


wherein the activation state of the precursor cell is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the isolated precursor cell as compared to the control is indicative of rheumatoid arthritis.
The phrase “diagnosing or monitoring rheumatoid arthritis” as used herein refers to a method or process of determining if a subject has or does not have rheumatoid arthritis, or determining the severity or degree of rheumatoid arthritis.
The term “subject” as used herein refers to any member of the animal kingdom, preferably a human being.
The term “sample” as used here refers to any fluid, cell or tissue sample from an individual which includes the precursor cell of the invention. In one embodiment, the sample is from the circulatory system of the individual.
The term “control” as used herein refers to a sample from a subject or a group of subjects who are either known as having rheumatoid arthritis or not having rheumatoid arthritis, or who are known as having a particular severity or degree of rheumatoid arthritis or not. A subject known not to have rheumatoid arthritis is also referred to as a “healthy individual” herein. For example, the control can be from a healthy individual, a subject with early stage rheumatoid arthritis or a subject with late stage rheumatoid arthritis. A person skilled in the art will appreciate that a subject with early stage rheumatoid arthritis can be defined to include individuals within the first year of onset of symptoms with 3 swollen joints.
The phrase “difference in number of isolated precursor cells in the sample from the subject as compared to the control is indicative of rheumatoid arthritis” refers to the difference in frequency of cells. There are generally greater numbers of the isolated precursor cells in samples from subjects with rheumatoid arthritis as compared to healthy individuals. In addition, there are greater numbers of the isolated precursor cells in samples from subjects as the disease progresses. For example, there are greater numbers of the isolated precursor cell from subjects with late stage rheumatoid arthritis as compared to subjects with early stage rheumatoid arthritis. Thus, if the control is a healthy individual, then there are greater numbers of the isolated precursor cells in the samples from subjects with rheumatoid arthritis as compared to the control. If the control is a subject with early stage rheumatoid arthritis, then there are greater numbers of the precursor cells in samples with late stage rheumatoid arthritis as compared to the control.
The “activation state of the isolated precursor cell” can be determined by measuring the activation status of signaling molecules within the isolated precursor cell. For example, the activation status of signaling molecules can be determined by measuring the phosphorylation levels of signaling molecules, such as STAT3, STAT5, ERK and/or p38 MAPK.
The phrase “difference in the activation state of the isolated precursor cell as compared to the control is indicative of rheumatoid arthritis” refers to a difference in the frequency or levels of phosphorylation of signaling molecules in the isolated precursor cell, including STAT3, STAT5, ERK and p38 MAPK, as compared to the control. There are more frequent and/or higher levels of phosphorylation of signaling molecules in samples from subjects with rheumatoid arthritis as compared to healthy individuals. There are generally more frequent and/or higher levels of phosphorylation of signaling molecules in samples from subjects as the disease progresses. For example, there are more frequent and/or higher levels of phosphorylation of signaling molecules in samples from subjects with late stage rheumatoid arthritis as compared to subjects with early stage rheumatoid arthritis. Thus, if the control is a healthy individual, then there are more frequent and/or higher levels of phosphorylation of signaling molecules in precursor cells in samples from subjects with rheumatoid arthritis as compared to the control. If the control is a subject with early stage rheumatoid arthritis, then there are more frequent and/or higher levels of phosphorylation of signaling molecules in precursor cells in samples from subjects with late stage rheumatoid arthritis as compared to the control.
The invention also includes the use of the isolated precursor cell of the invention to diagnose or monitor rheumatoid arthritis.
The isolated precursor cell of the invention can also be used in methods of drug discovery or methods to identify substances that can treat or prevent rheumatoid arthritis. For example, an additional aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the number of isolated precursor cells of the invention in a sample from a subject treated with a substance; and
        (b) comparing the number of isolated precursor cells from the sample with a control;
    
    


wherein a difference in the number of isolated precursor cells in the sample from the subject as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
In another example, the invention includes a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the activation state of the isolated precursor cell of the invention from a sample from a subject treated with a substance; and
        (b) comparing the activation state of the isolated precursor cell from the sample with a control;
    
    


wherein the activation state of the precursor cell is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the isolated precursor cell as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
The phrase “treat or prevent rheumatoid arthritis” as used herein refers to a medical aid to counteract the disease itself, the symptoms and/or the progression of the disease.
“Measuring the phosphorylation levels of signaling molecules” as used herein refers to measuring the frequency and/or intensity of phosphorylation of signaling molecules, such as STAT3, STAT5, ERK and/or p38 MAPK.
A person skilled in the art will appreciate that the control can be a sample from a subject not treated with a substance or treated with a substance that is known not to treat or prevent rheumatoid arthritis. In one embodiment, reduced numbers of the isolated precursor cell in the sample as compared to the control is indicative of a substance for the treatment or prevention of rheumatoid arthritis. In another embodiment, a reduced activation state of the isolated precursor cell in the sample as compared to the control is indicative of a substance for the treatment or prevention of rheumatoid arthritis. In addition, the control can be a sample from the same subject, but before treatment with the substance to be tested or samples from the subject taken at different points of time during treatment with the substance to be tested.
Substances for the treatment or prevention of rheumatoid arthritis can also be identified using cells or cell lines. For example, individual precursor cells or cell lines derived from the precursor cell of the invention can be contacted with a substance and then the activation state of the cells can be compared to a control.
The inventors have also studied the activation status of circulating CD3+ cells from rheumatoid arthritis patients at different stages of disease. They discovered that there are progressive increases in the phosphorylation of signaling molecules, such as STAT3 and p38 MAPK, in CD3+ cells, which correlate with disease progression.
Accordingly, the invention includes method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the activation state of a CD3+ cell from a sample from the subject; and
        (a) comparing the activation state of the CD3+ cell from the sample with a control;
    
    


wherein, the activation state of the CD3+ cell is determined by measuring the phosphorylation levels of signaling molecules, such as STAT3 and/or p38 MAPK, and
wherein a difference in the activation state of the CD3+ cell as compared to the control is indicative of rheumatoid arthritis.
The “activation state of the CD3+ cell” can be determined by measuring the activation status of signaling molecules within CD3+ cell. For example, the activation status of signaling molecules can be determined by measuring the phosphorylation levels of signaling molecules, such as STAT3 and/or p38 MAPK.
The phrase “difference in the activation state of the CD3+ cell as compared to the control” refers to a difference in the frequency or levels of phosphorylation of signaling molecules in the CD3+ cell, including STAT3 and/or p38 MAPK, as compared to the control.
The term “sample” as used here refers to any fluid, cell or tissue sample from an individual which includes a CD3+ cell.
The invention also includes the use of a CD3+ cell to diagnose or monitor rheumatoid arthritis in a subject.
The findings of the inventors can also be used in methods of drug discovery. Accordingly, the invention includes method of identifying a substance to treat or prevent rheumatoid arthritis comprising the steps:

    
    
        (a) determining the activation state of a CD3+ cell from a sample from a subject treated with a substance; and
        (b) comparing the activation state of the CD3+ cell from the sample with a control;
    
    


wherein the activation state of the CD3+ cell is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the CD3+ cell as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
The invention also includes the use of a CD3+ cell to identify a substance to treat or prevent rheumatoid arthritis.
The inventors have also analyzed and characterized the gene expression and protein expression profiles of synovial tissue fibroblast-like synovial cells from subjects with rheumatoid arthritis and osteoarthritis. The inventors have discovered that there are different gene expression and protein expression profiles, and protein phosphorylation profiles in synovial tissue fibroblast-like synovial cells from subjects with rheumatoid arthritis, osteoarthritis and healthy individuals.
The inventors discovered a number of rheumatoid arthritis specific genes that can be used to characterize the gene expression profile in the method of the invention. These genes include transport, apoptosis regulatory, cell adhesion, cell surface signaling receptors, intracellular signaling, secreted stimulatory and immunomodulatory genes. In addition, the inventors discovered a significant differential expression of 154 genes in fibroblast-like synovial cells in subjects with osteoarthritis or rheumatoid arthritis. See Tables 1 and 2.
Accordingly, the invention includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the gene expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the gene expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the gene expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of rheumatoid arthritis.
In one embodiment, the gene expression profile that characterizes subjects with rheumatoid arthritis includes enhanced gene expression of one or more of the genes listed in Table 1.
In another embodiment, the gene expression profile that characterizes subjects with rheumatoid arthritis includes enhanced gene expression of genes encoding immunoglobulin constant regions, CD53, CD11b, CD18, CD86, CD206, CD163, mannose receptor, DC-SIGN, C3AR1, Fc-receptors, complement receptors, and/or MHC class II molecules as compared to the control.
The invention also includes a method of diagnosing or monitoring osteoarthritis in a subject, comprising the steps:

    
    
        (a) determining the gene expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the gene expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the gene expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of osteoarthritis.
The term “control” as used here refers to a sample from a subject or group of subjects who are either known as having osteoarthritis or not, or who are known as having a particular severity or degree of osteoarthritis or not.
In one embodiment, the gene expression profile that characterizes subjects with osteoarthritis includes enhanced gene expression of one or more of the genes listed in Table 2.
The term “sample” as used here refers to any fluid, cell or tissue sample from an individual which includes a synovial tissue fibroblast-like synovial cell.
The term “gene expression profile” as used herein refers to the level of RNA expressed from one or more gene in the synovial tissue fibroblast-like synovial cell from a sample from the subject.
The term “difference in gene expression profile” as used here refers to an increase or decrease in the measurable expression of RNA of a particular gene or group of genes as compared to the measurable expression of RNA of the same gene or group of genes in a second sample. The comparison can be made between individual samples or populations of samples. In one embodiment, the differential expression can be compared using the ratio of the level of expression of the gene as compared with the expression level of the gene of a control, wherein the ratio is not equal to 1.0. For example, an RNA is differentially expressed if the ratio of the level of expression in a first sample as compared with a second sample is greater than or less than 1.0. For example, a ratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.001 or less. In another embodiment the differential expression is measured using p-value. For instance, when using p-value, a gene is identified as being differentially expressed as between a first and second population when the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.01, even more preferably less than 0.005, the most preferably less than 0.001.
In addition to measuring the gene expression profile, a person skilled in the art will appreciate that the protein expression profile of a synovial tissue fibroblast-like synovial cell can be measured.
The term “protein expression profile” as used herein refers to the level of one or more proteins expressed in the synovial tissue fibroblast-like synovial cell from a sample from the subject. The protein expression profile can include measurements of the expression of transport, apoptosis regulatory, cell adhesion, cell surface signaling receptors, intracellular signaling, secreted stimulatory and immunomodulatory proteins. This includes measuring the protein expression of immunoglobulin constant regions, CD53, CD11b, CD18, CD86, CD206, CD163, mannose receptor, DC-SIGN, C3AR1, Fc-receptors, complement receptors, and/or MHC class II molecules.
Accordingly, the invention includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the protein expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the protein expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of rheumatoid arthritis.
In one embodiment, the protein expression profile that characterizes subjects with rheumatoid arthritis includes enhanced protein expression of one or more proteins encoded by the genes listed in Table 1.
The invention also includes a method of diagnosing or monitoring osteoarthritis in a subject, comprising the steps:

    
    
        (a) determining the protein expression profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the protein expression profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein expression profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of osteoarthritis.
In one embodiment, the protein expression profile that characterizes subjects with osteoarthritis includes enhanced protein expression of one or more of the proteins encoded by the genes listed in Table 2.
The term “difference in protein expression profile” as used here refers to an increase or decrease in the measurable expression of a particular protein or group of proteins as compared to the measurable expression of the same protein or group of proteins in a second sample. The comparison can be made between individual samples or populations of samples. In one embodiment, the differential expression can be compared using the ratio of the level of expression of the protein as compared with the expression level of the protein of a control, wherein the ratio is not equal to 1.0. For example, a protein is differentially expressed if the ratio of the level of expression in a first sample as compared with a second sample is greater than or less than 1.0. For example, a ratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.001 or less. In another embodiment the differential expression is measured using p-value. For instance, when using p-value, a protein is identified as being differentially expressed as between a first and second population when the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.01, even more preferably less than 0.005, the most preferably less than 0.001.
In addition to gene and protein expression profiles, the inventors also examined protein phosphorylation profiles in synovial tissue fibroblast-like synovial cells.
Accordingly, the invention includes a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the protein phosphorylation profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (a) comparing the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of rheumatoid arthritis.
The term “protein phosphorylation profile” as used herein refers to the level or frequency of phosphorylation of one or more proteins expressed in synovial tissue fibroblast-like synovial cells from a sample from a subject.
In one embodiment, the difference in the protein phosphorylation profile includes a difference in phosphorylation of AKT, FAK, p38, JNK, cdc-2 and/or PLC-g1 in the synovial tissue fibroblast-like synovial cells in the subject as compared to the control.
The invention also includes a method of diagnosing or monitoring osteoarthritis in a subject, comprising the steps:

    
    
        (a) determining the protein phosphorylation profile of a synovial tissue fibroblast-like synovial cell from a sample from the subject; and
        (b) comparing the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell from the sample with a control;
    
    


wherein a difference in the protein phosphorylation profile of the synovial tissue fibroblast-like synovial cell as compared to the control is indicative of osteoarthritis.
The methods of the invention can also be used to identify substances to treat or prevent rheumatoid arthritis or osteoarthritis.
The inventors also determined that the number of circulating fibrocytes and the activation status of fibrocytes can be used to diagnose or monitor rheumatoid arthritis in a subject.
The term “sample” as used here refers to any fluid, cell or tissue sample from an individual which includes circulating fibrocytes. In one embodiment, the sample is from the circulatory system of the individual.
Accordingly, a further aspect of the invention is a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the number of circulating fibrocytes in a sample from the subject; and
        (b) comparing the number of fibrocytes from the sample with a control;
    
    


wherein a difference in the number of fibrocytes in the sample from the subject as compared to the control is indicative of rheumatoid arthritis.
The phrase “difference in number of fibrocytes in the sample from the subject as compared to the control is indicative of rheumatoid arthritis” refers to the difference in frequency of cells. There are generally greater numbers of the circulating fibrocytes in samples from subjects with rheumatoid arthritis as compared to healthy individuals. In addition, there are greater numbers of circulating fibrocytes in samples from subjects as the disease progresses. For example, there are greater numbers of circulating fibrocytes from subjects with late stage rheumatoid arthritis as compared to subjects with early stage rheumatoid arthritis. Thus, if the control is a healthy individual, then there are greater numbers of circulating in the samples from subjects with rheumatoid arthritis as compared to the control. If the control is a subject with early stage rheumatoid arthritis, then there are greater numbers of circulating fibrocytes in samples with late stage rheumatoid arthritis as compared to the control.
Another aspect of the invention, is a method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:

    
    
        (a) determining the activation state of a circulating fibrocyte from a sample from the subject; and
        (b) comparing the activation state of the fibrocyte from the sample with a control;
    
    


wherein the activation state of the fibrocyte is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the fibrocyte as compared to the control is indicative of rheumatoid arthritis.
The “activation state of the fibrocyte” can be determined by measuring the activation status of signaling molecules within the isolated precursor cell. For example, the activation status of signaling molecules can be determined by measuring the phosphorylation levels of signaling molecules, such as STAT5.
The phrase “difference in the activation state of the fibrocyte as compared to the control is indicative of rheumatoid arthritis” refers to a difference in the frequency or levels of phosphorylation of signaling molecules in the isolated precursor cell, such as STAT5, as compared to the control. There are more frequent and/or higher levels of phosphorylation of signaling molecules in samples from subjects with rheumatoid arthritis as compared to healthy individuals. There are generally more frequent and/or higher levels of phosphorylation of signaling molecules in samples from subjects as the disease progresses. For example, there are more frequent and/or higher levels of phosphorylation of signaling molecules in samples from subjects with late stage rheumatoid arthritis as compared to subjects with early stage rheumatoid arthritis. Thus, if the control is a healthy individual, then there are more frequent and/or higher levels of phosphorylation of signaling molecules in precursor cells in samples from subjects with rheumatoid arthritis as compared to the control. If the control is a subject with early stage rheumatoid arthritis, then there are more frequent and/or higher levels of phosphorylation of signaling molecules in precursor cells in samples from subjects with late stage rheumatoid arthritis as compared to the control.
The invention also includes using circulating fibrocytes to diagnose or monitor rheumatoid arthritis.
An additional aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the number of circulating fibrocytes in a sample from a subject treated with a substance; and
        (b) comparing the number of fibrocytes from the sample with a control;
    
    


wherein a difference in the number of fibrocytes in the sample from the subject as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
A further aspect of the invention is a method of identifying a substance to treat or prevent rheumatoid arthritis, comprising the steps:

    
    
        (a) determining the activation state of circulating fibrocytes from a sample from a subject treated with a substance; and
        (b) comparing the activation state of the fibrocyte from the sample with a control;
    
    


wherein the activation state of the fibrocyte is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a difference in the activation state of the fibrocyte as compared to the control is indicative of a substance to treat or prevent rheumatoid arthritis.
In addition, the invention includes the use of circulating fibrocytes to identify a substance to treat or prevent rheumatoid arthritis.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1
Characterization of Gene and Protein Expression Profiles in RA ST FLS Cells
The activated phenotype of distinct cell populations in affected RA patients determines disease severity. Suppression of the activated phenotype of cells in early RA synovitis will subdue the disease process. A clear understanding of those factors that contribute to the activated phenotype is required. The objective of the inventors' studies was to determine the signature gene and protein expression profiles in target cell populations in patients diagnosed with RA. The inventors' hypothesized that a signature pattern of activated factors in distinct cell types would identify potential therapeutic targets. Over the past few years, the inventors have initiated a collection of blood, synovial fluid and ST from RA patients, osteoarthritis (OA) patients and trauma patients at the time of joint surgery. Sample collection involves confirmation of the diagnosis of RA/OA using clinical, serologic and radiologic data and informed consent on all study participants. Clinical parameters are recorded at the time of sample collection. Synovial samples are collected by joint aspiration, fine needle biopsy and via arthroscopic or other surgeries. FLS cells from ST from affected RA joints, OA joints and tissue from trauma patients (non-RA, non-OA, surgery) were collected using a negative-selection protocol for cell fractionation, and cultured in DMEM media. Initial studies examined gene expression profiles of RNA from freshly harvested ST, cells that were passaged up to 3× and cells maintained beyond three passages. Affymetrix microarray analysis (U133 Plus 2.0 microarray representing over 30,000 genes) was performed. RA FLS cells at 3× passage exhibit similar gene expression profiles compared to freshly harvested FLS cells, yet the inventors observed that this characteristic gene expression profile changed with extended time in culture. All subsequent gene expression analyses were conducted on RNA extracted from ST FLS cells cultured for <3 passages. Gene expression data were initially analyzed using GeneSpring 6.1 software (Silicon Genetics). Results of gene tree data analysis of ST FLS cells have revealed distinct and reproducible expression profiles, reflective of the different patient populations (trauma patients, OA, RA), that is significant as determined by one-way ANOVA. Gene tree data analysis sorted the RA-specific genes into functional groupings: transport, apoptosis regulatory, cell adhesion, cell surface signaling receptors, intracellular signaling, secreted stimulatory, and immunomodulatory genes. A gene tree based comparison of OA versus RA ST FLS cell expression profiles revealed significant differential expression of 154 genes (one-way ANOVA, p<0.01), including 17 apoptosis regulatory, 17 cell adhesion, 39 cell surface receptors, 25 immunomodulatory and 28 signal transduction genes. To further analyze these data, the Binary Tree-Structured Vector Quantization (BTSVQ) program was used (11). BTSVQ is a computational tool that combines partitive k-means clustering and Self-Organizing Maps (SOM) to analyze and visualize microarray gene expression data with minimal noise and without pre-conceived bias. These analyses confirmed distinct signature patterns of gene expression for FLS cells derived from RA (n=16) and OA (n=20) specimens and further distinguished healthy control (trauma patients, n=6) gene expression patterns from RA and OA expression profiles (FIG. 1). Differentially regulated genes in RA FLS cells included cytokines, chemokines and receptors. In agreement with previous studies (12), the inventors also show a transformed phenotype in RA FLS cells, with expression of many genes that are typically not found in fibroblasts. High levels of gene expression for immunoglobulin constant regions: IGKC, IGLC2 and IGHM were seen, typically expressed in B cells. Also, gene expression for many receptors found on antigen presenting cells such as: CD53, CD11b, CD18, CD86, scavenger receptors (CD206, CD163 and the mannose receptor, MRC), DC-SIGN and complement receptor (C3AR1), were observed. The MRC processes molecules for presentation of MHCII and binds to both endogenous and exogenous ligands. Increases in gene expression for Fc- and complement receptor gene expression were observed in RA FLS cells and suggest that these cells trap antigens in the form of immune complexes on their cell surface for presentation. Gene expression for HLA-DR, DQ and DP were also upregulated in RA vs. OA or control FLS cells in the array analyses. HLA molecules are expressed on CD68+ synoviocytes following treatment with IFN-γ (13). Notably, gene expression for CD68 was not evident in the isolated FLS cells. Upregulation of HLA molecules would be consistent with the role of synovial fibroblasts in antigen presentation during an autoimmune disease. The likelihood of contaminating leukocytes, e.g. macrophage-like synoviocytes (MLS), in the fibroblast preparations was eliminated since only adherent prolyl-4 hydroxylase (5B5 antibody) positive cells were processed for gene expression analysis, and there was no significant CD2, CD3 or CD5 gene expression observed in the analysis. This suggests that RA FLS cells either upregulate the B cell/macrophage/dendritic cell markers, de-differentiate into this phenotype, or that immature fibroblast-like cells are recruited and differentiate into these cells in affected joints.
In parallel, protein extracted from the RA, OA and surgical trauma ST FLS cells was analyzed using the BD BioSciences PowerBlot Western immunoarray technology platform. The full array allows 996 proteins to be interrogated, and the inventors have developed a customized mini-array that specifically interrogates phospho-proteins. In agreement with the data for distinct gene expression, distinct protein expression patterns were observed in the RA FLS cells compared to OA FLS cells (FIG. 2). RA FLS cells were activated as determined by higher levels of phospho-AKT, phospho-FAK, phospho-p38, JNK, cdc-2 and PLC-γ1 proteins (FIG. 3). The PI3K/AKT pathway plays an important role in balancing apoptosis and survival (14) and is involved in cell cycle progression, glucose metabolism and chemotaxis (15). RA fibroblasts show enhanced survival in response to TNF-a (16) and TGF-β (17) through an AKT-dependent pathway (17). Additionally, this pathway has also been implicated in TRAIL induced proliferation in RA fibroblasts (18). Activation of the insulin-like growth factor-1 receptor (IGF-1R) (19), IL-17 (20) and the IL-18 receptor (21) also effect signal transduction in an AKT-dependent manner to induce cytokine/chemokine or adhesion molecule synthesis in RA fibroblasts. Focal adhesion kinase (FAK) regulates anti-apoptotic pathways through PI3K-dependent and -independent pathways. FAK is a widely expressed cytoplasmic protein tyrosine kinase involved in integrin mediated signal transduction. B1 integrin signaling through FAK upregulates ICAM-1 and Fas in RA FLS cells (22). The JNK and p38 pathways are preferentially activated by stress, inflammatory cytokines and growth factors. TNF can signal through both JNK and p38 to activate AP-1 and NF-kB (23) and IL-1 upregulates MMP13 (24) and collagenase 1 (25) via the JNK pathway in RA FLS cells. The entry of all cells into mitosis is regulated by cdc2 and increased cdc2 phosphorylation has been reported in RA fibroblasts and may be involved in aberrant mitosis (26). Viewed altogether, the data confirms that multiple genes and signaling pathways are activated in RA FLS cells contributing to the distinct phenotype of these cells.
Example 2
Fibrocytes in RA
RA is a complex heterogeneous disease. Hyperproliferation of FLS cells is considered to be a major contributor to pannus formation; however, little direct evidence supports hyperproliferation of these cells in vivo. Few mitotic figures are observed, thymidine uptake only occurs in a percentage of the cells (27) and FLS cells divide very slowly in culture (28). Decreased senescence in FLS cells may occur and RA FLS cells have been shown to retain telomerase activity (29), but these cells are not immortalized in vitro (29) (30). The outgrowth of FLS cells in the ST of affected RA joints is, therefore an enigma, and one possible explanation might be that FLS cells are recruited from the circulation. Many years ago, a circulating population of fibroblasts-like cells, was identified (31). More recently, these cells have been characterized further, designated as fibrocytes, and implicated in influencing disease development in tumor biology, scleroderma, asthma and pulmonary fibrosis (32) (33) (34) (35).
Fibrocytes are unique circulating cells that are relatively rare in the circulation, comprising only 0.1-1% of the white blood cells. Characterization of these cells has been predominantly by FACS staining of cells cultured for up to two weeks ex vivo and relatively little work has been performed in vivo. In vitro, fibrocytes have a unique phenotype and express markers of both stromal cells and hematopoietic cells, including: fibronectin, collagen, prolyl 4-hydroxylase, CD11a, CD11b, CD13, CD18, CD45RO, ICAM1, CD80, CD86, CXCR4, CCR7 and CCR5 (32) (36). Fibrocytes originate from the bone marrow (37) and arise from a CD14+ve pool of cells in the circulation, but fibrocytes themselves become CD14− (38). At the time of culture, these cells express CD34 and CD45 (33). When cultured ex vivo, these cells become adherent and develop a spindle-shaped morphology (33) (39) and downregulate CD45 and CD34. Three weeks after culture these cells express the myofibroblast marker, a-smooth muscle actin (SMA) (33), and have the ability to contract collagen gels in vitro (38). Ex vivo cultured fibrocytes differentiated into a-SMA expressing cells when directly co-cultured with T cells or upon TGF-b stimulation (38). Fibrocyte differentiation is inhibited by serum amyloid P (SAP) (36). Recently, in vivo evidence indicates that the fibrocyte population contributes to the myofibroblast population in a murine wound healing model (37). Fibrocytes secrete collagen and fibronectin, thereby contributing to granulation formation, and they are contractile, enhancing wound contraction and healing. They have also been shown to present antigen (40) and can secrete chemokines, cytokines and angiogenic factors (32), suggesting that fibrocytes contribute to both inflammation and its resolution.
Increased myofibroblast-like cells in the joint are observed in RA and correlate with the degree of inflammatory synovitis. It has been suggested that RA myofibroblasts originate from the circulating fibrocyte population (41) (42). The percentage of a-SMA expressing cells in RA patients varies from 1-30% (43) and the inventors have observed upregulated a-SMA gene expression in RA FLS cells (FIG. 1). Whether these myofibroblasts were derived locally, or were recruited from the circulating pool, is unclear. Certainly, circulating endothelial progenitor cells are increased during the onset of collagen induced arthritis, likely contributing to neoangiogenesis in affected tissues (Kurosaka et al 2005). Endothelial precursors are recruited in RA (44) and increased circulating endothelial precursors are observed in murine CIA (45) and in patients with RA (46). The inference is that circulating endothelial precursors will traffic to affected RA joints to promote the neoangiogenesis. Given that fibrocytes express CCR3, CCR5 and CCR7, and the cognate ligands for these receptors are found in synovial fluid, it is likely that fibrocytes will also traffic to the inflamed RA joint. Indeed, RA FLS cells expressed higher levels of some fibrocyte markers including: CCR5, CXCR4, CD54, CD18, CD11b and CD45 (FIG. 1). Fibrocytes and fibroblasts can differentiate into a-SMA expressing myofibroblasts upon stimulation with TGF-b Notably, increased levels of TGF-b are present in RA STs. Myofibroblast transformation involves activation of adhesion and integrin signaling through FAK and AKT pathways (47). TGF-b promotes AKT-dependent survival of mesenchymal cells through p38 MAPK-induced growth factor secretion (48). Constitutive phosphorylation of FAK is involved in myofibroblast differentiation in scleroderma (49). Apparently, many of the pathways the inventors have identified as potentially activated in the RA FLS cells have been implicated in fibrocyte differentiation.
Example 3
Signal Taxonomy in Defined RA Cell Populations
Changes in intracellular protein levels, subcellular localization, or activation state are considered to be reflective of a cell's capabilities or functions. Some of these events are relatively transitory—such as some phosphorylation of proteins in cell signaling cascades. Some of the relevant cell populations are so rare as to make their isolation for standard biochemical analysis nearly impossible. Remodeling of such cell signaling mechanisms drives disease pathogenesis contributing to immune cell dysregulation despite intense therapy regimens. Therefore, to understand how signaling networks are remodeled in RA there is a need to measure complex populations of immune system cells and phenotype them not only for their cell lineage status, but also for their relative activation state.
Studies in human myeloid leukemia have shown that signaling can be mapped at the individual cell level by flow cytometry and have demonstrated links between oncogene mutations and patterns of proliferative signaling in tumors (50). Furthermore, this work suggested that a tumor could be described by its signal transduction potential and that this status stratified patient risk of relapse following chemotherapy. Since RA is a systemic and chronic inflammatory autoimmune disease that targets synovial joints, disease pathogenesis is multifactorial and extends beyond T or FLS cell mediated destruction of cartilage. Production of pro-inflammatory cytokines such as TNFa and IL-1β by activated monocytes and macrophages contribute to tissue destruction by activation of chondrocytes and fibroblasts that release metalloproteinases and collagenases into the synovial cavity. Cartilage loss and bone erosion are physical manifestations of disease progression. Additional blood borne compartments, such as B cells, contribute to production of autoantibodies and rheumatoid factor and have been regarded as playing important roles. Both T and B cell lymphocyte deregulation, as well as the involvement of chondrocytes and fibroblasts suggest a dynamic interaction of cell-to-cell communications contribute to disease pathogenesis of RA.
The nature of the intracellular pathways activated with most primary immune cell interactions is not well understood in vivo and less clear in disease states. In many cases it is only understood using derived cell lines, in vitro, and at best is often accomplished by lysis of cells and western immunoblots of total cellular material. Therefore significant information on population variations that exist is missed and advances in genomics and proteomic technologies that rely on lysate material do not access the heterogeneous subsets that exist in the immune system.
Multiparameter flow cytometric analysis allows for small subpopulations to be discerned using cell surface markers—representing different cellular subsets, differentiation or activation states.
The inventors developed a series of assay systems for flow cytometric based biochemical analysis at the single cell level for kinase and phospho-protein profiling. Measurement of up to 12 simultaneous protein or kinase events per cell for studying signaling events in primary cells are possible with these systems (51) (52) (53) (54). This allows for unprecedented study of signaling in autoimmune diseases as immunocytes are particularly amenable to these techniques.
Underlying the gross dysfunctionality of autoimmunity are the signaling systems that drive their actions. The last two decades of research have uncovered numerous pathways leading from surface receptors to gene regulation. Many of these pathways, if not all, at one point or another pass signals through phosphorylation or dephosphorylation events on proteins or lipids. Integration of signaling events leads to relocalization of proteins within cells, such as with translocation of proteins to the nucleus. Until recently, measurement of such events has been limited to cell lines or bulk lysis assays. Interpretation of such assays is considered a pale rendition of what we know is the intricacy of complex population primary cell events. Therefore a true understanding of the nature of signaling dysfunctions during disease processes in heterogeneous patient samples or animal model systems has been beyond reach.
It can be hypothesized that the activation profiles of proteins, such as phospho-proteins that drive proliferation and activation signaling cascades will differ, in disease states, both from a “normal” profile of a non-disease presence and from other samples with significantly different pathology. During disease pathogenesis, cells are accessing different environmental cues, or ignoring those that might be attempting to block their replication (or induce their apoptosis). As such, it would be expected that there would be underlying differences in the activation profiles of certain phospho-proteins across immune and non-immune cells in RA pathogenesis.
Therefore, with sufficient understanding of relationships between cell signaling and immunopathology, activation profiles of phospho-proteins could indicate the presence of individual aggressive cell subsets within a complex population of cells. Since it is hard to predict which kinases might be relevant in different cell subsets, this hypothesis has limited utility in the absence of a high throughput manner to measure many kinases. However, if one could measure dozens of kinase activation profiles—or their target proteins—simultaneously—there is the opportunity to generate invaluable information about the role of signaling events in rheumatologic disease, test hypotheses of signaling systems in response to various therapies, as well as develop diagnostic indicators based on kinase profiles. Finally, it might be possible to reveal signaling states that are hidden from obvious view if one were only looking at basal phosphorylation states.
Knowledge of intracellular signaling differences among arthritic immune cells could therefore provide the basis for an improved autoimmune classification system. Importantly, such a classification system would go beyond a simple signature, but could be used to infer mechanism associated with the signature.
In the present studies the inventors have shown that by surveying phospho-protein/basal phosphorylation states, the underlying dysregulated signaling nodes in primary human RA samples can be revealed and the identification of signaling pathology profiles can be enabled. Specifically, based on our gene and protein expression data elaborating an emerging phenotype for the RA FLS, the inventors examined peripheral blood mononuclear cells (PBMC) for a fibrocyte population with similar properties. At the outset, PBMC from healthy individuals were analyzed by multiparameter flow cytometry to identify the fibrocyte population. The data in FIG. 4 indicate that immunostaining for collagen, CD34, CD45, prolyl 4-hydroxylase and CD14 identifies this precursor fibrocyte population. In subsequent experiments, multiparametric staining using both surface and intracellular stains was employed to determine the activation status of the circulating fibrocytes in the PBMC of healthy individuals, patients with early RA and later stage disease. Early RA was defined as patients within the first year of onset of symptoms with 3 swollen joints. Changes in the signaling status that occurs during the functional activation of this fibrocyte population might include phosphorylation-activation of STATs, Erk and MAP kinases. Accordingly, using polychromatic analyses the inventors examined the frequency of activated fibrocytes, focusing on STAT3, STAT5, Erk and p38 MAPK. CD3-CD45+collagen+ fibrocytes were stained for phospho-STAT3 (FIG. 5), phospho-STAT5 (FIG. 6), phospho-Erk (FIG. 7) and phospho-p38 MAPK (FIG. 8) and the frequency of staining determined in each patient population: healthy individuals (n=20), early RA patients (n=8), late stage disease RA patients (n=4). These data indicate that the frequency of phospho-staining for these signaling effectors in the RA patient fibrocytes is significantly higher (panels B) than that seen in fibrocytes from healthy individuals.
Example 4
Collagen-Induced Arthritis Model
Collagen-induced arthritis (CIA) is a widely used model of rheumatoid arthritis (55) (56). Mice with CIA exhibit obvious swelling of paws and joints as compared to control animals (FIG. 12A). The mice were given a clinical disease score based on the severity of the disease (FIG. 12B). FIG. 12C shows immunohistochemistry staining of the joint.
The inventors collected PBMC by cardiac puncture at different stages of disease and analyzed the cells by FACS for positive staining to α-SMA and collagen. As can be seen in FIGS. 12D and E, the number of circulating fibrocytes is higher in mice with higher disease scores.
The circulating fibrocytes also showed increased p-STAT5 in the animals with CIA as compared to the control animals (FIG. 13).
FIGS. 14 and 15 show the results of immunohistochemistry of samples from mice with CIA as compared to controls.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.













TABLE 1





Probe set
Public ID

Chromosomal





ID number
number (NCBI)
Target description
location
Unigene ID
Gene Title
Gene Symbol







209765_at
Y13786
gb: Y13786.2 /DB_XREF = gi: 12053590 /FEA = FLmRNA /CNT = 58
chr5q32-q33
Hs.289368
ADAM metallopeptidase domain
ADAM19




/TID = Hs.278679.1 /TIER = Stack /STK = 24 /UG = Hs.278679 /LL = 8728


19 (meltrin beta)




/UG_GENE = ADAM19 /DEF = Homo sapiens mRNA for meltrin-betaADAM 19




homologue. /PROD = meltrin-betaADAM 19 homologue /FL = gb: AF311317.1


206134_at
NM_014479
gb: NM_014479.1 /DB_XREF = gi: 7657318 /GEN = M12.219 /FEA = FLmRNA
chr8p21.2
Hs.145296
ADAM-like, decysin 1
ADAMDEC1




/CNT = 21 /TID = Hs.145296.0 /TIER = FL + Stack /STK = 14 /UG = Hs.145296




/LL = 27299 /DEF = Homo sapiens disintegrin protease (M12.219), mRNA.




/PROD = disintegrin protease /FL = gb: NM_014479.1


206513_at
NM_004833
gb: NM_004833.1 /DB_XREF = gi: 4757733 /GEN = AIM2 /FEA = FLmRNA
chr1q22
Hs.105115
absent in melanoma 2
AIM2




/CNT = 15 /TID = Hs.105115.0 /TIER = FL /STK = 7 /UG = Hs.105115 /LL = 9447




/DEF = Homo sapiens absent in melanoma 2 (AIM2), mRNA. /PROD = absent




in melanoma 2 /FL = gb: AF024714.1 gb: NM_004833.1


218332_at
NM_018476
gb: NM_018476.1 /DB_XREF = gi: 8923715 /GEN = HBEX2 /FEA = FLmRNA
chrxq21-q23
Hs.334370
brain expressed, X-linked 1
BEX1




/CNT = 116 /TID = Hs.283719.0 /TIER = FL + Stack /STK = 54 /UG = Hs.283719




/LL = 55859 /DEF = Homo sapiens uncharacterized hypothalamus protein




HBEX2 (HBEX2), mRNA. /PROD = uncharacterized hypothalamus protein




HBEX2


205715_at
NM_004334
gb: NM_004334.1 /DB_XREF = gi: 4757873 /GEN = BST1 /FEA = FLmRNA
chr4p15
Hs.169998
bone marrow stromal cell antigen 1
BST1




/CNT = 43 /TID = Hs.169998.0 /TIER = FL + Stack /STK = 21 /UG = Hs.169998




/LL = 683 /DEF = Homo sapiens bone marrow stromal cell antigen 1 (BST1),




mRNA. /PROD = bone marrow stromal cell antigen 1 precursor /FL = gb: D21


202953_at
NM_000491
gb: NM_000491.2 /DB_XREF = gi: 11038661 /GEN = C1QB /FEA = FLmRNA
chr1p36.3-p34.1
Hs.8986
complement component 1, q
C1QB




/CNT = 135 /TID = Hs.8986.0 /TIER = FL + Stack /STK = 16 /UG = Hs.8986 /LL = 713


subcomponent, beta polypeptide




/DEF = Homo sapiens complement component 1, q subcomponent, beta




polypeptide (C1QB), mRNA. /PROD = complement component 1, q




subcompon


209906_at
U62027
gb: U62027.1 /DB_XREF = gi: 1511643 /GEN = HNFAG09 /FEA = FLmRNA
chr12p13.31
Hs.155935
complement component 3a
C3AR1




/CNT = 43 /TID = Hs.155935.0 /TIER = FL + Stack /STK = 19 /UG = Hs.155935


receptor 1




/LL = 719 /DEF = Human anaphylatoxin C3a receptor (HNFAG09) mRNA,




complete cds. /PROD = anaphylatoxin C3a receptor /FL = gb: U62027.1




gb: U28488.1


229824_at
AL133706
gb: AL133706 /DB_XREF = gi: 6601894 /DB_XREF = DKFZp761O0410_s1
chr9q22.1-q22.2
Hs.4257
Endothelial differentiation,
C9orf47




/CLONE = DKFZp761O0410 /FEA = EST /CNT = 29 /TID = Hs.4257.0


sphingolipid G-protein-coupled




/TIER = Stack /STK = 28 /UG = Hs.4257 /UG_TITLE = ESTs


receptor, 3


206407_s_at
NM_005408
gb: NM_005408.1 /DB_XREF = gi: 4885586 /GEN = SCYA13 /FEA = FLmRNA
chr17q11.2
Hs.414629
chemokine (C—C motif) ligand 13
CCL13




/CNT = 19 /TID = Hs.11383.0 /TIER = FL /STK = 6 /UG = Hs.11383 /LL = 6357




/DEF = Homo sapiens small inducible cytokine subfamily A (Cys-Cys),




member 13 (SCYA13), mRNA. /PROD = small inducible cytokine subfamily A


209924_at
AB000221
gb: AB000221.1 /DB_XREF = gi: 2289718 /GEN = PARC /FEA = FLmRNA
chr17q11.2
Hs.16530
chemokine (C—C motif) ligand 18
CCL18




/CNT = 50 /TID = Hs.16530.0 /TIER = FL + Stack /STK = 21 /UG = Hs.16530


(pulmonary and activation-




/LL = 6362 /DEF = Homo sapiens mRNA for CC chemokine, complete cds.


regulated)




/PROD = CC chemokine /FL = gb: AB000221.1 gb: NM_002988.1


216598_s_at
S69738
gb: S69738.1 /DB_XREF = gi: 545464 /GEN = MCP-1 /FEA = mRNA /CNT = 1
chr17q11.2-q21.1
Hs.303649
chemokine (C—C motif) ligand 2
CCL2




/TID = Hs.303649.1 /TIER = ConsEnd /STK = 0 /UG = Hs.303649 /LL = 6347




/DEF = MCP-1 = monocyte chemotactic protein (human, aortic endothelial cells,




mRNA, 661 nt). /PROD = MCP-1


205114_s_at
NM_002983
gb: NM_002983.1 /DB_XREF = gi: 4506842 /GEN = SCYA3 /FEA = FLmRNA
chr17q21.1
Hs.512304
chemokine (C—C motif) ligand 3
CCL3 ///




/CNT = 78 /TID = Hs.73817.0 /TIER = FL + Stack /STK = 30 /UG = Hs.73817


/// chemokine (C—C motif) ligand 3-
CCL3L1 ///




/LL = 6348 /DEF = Homo sapiens small inducible cytokine A3 (homologous to


like 1 /// chemokine (C—C motif)
CCL3L3




mouse Mip-1a) (SCYA3), mRNA. /PROD = small inducible cytokine A3 (hom


ligand 3-like 3


214038_at
AI984980
gb: AI984980 /DB_XREF = gi: 5812257 /DB_XREF = wr88g11.x1
chr17q11.2
Hs.271387
chemokine (C—C motif) ligand 8
CCL8




/CLONE = IMAGE: 2494820 /FEA = FLmRNA /CNT = 29 /TID = Hs.271387.0




/TIER = Stack /STK = 16 /UG = Hs.271387 /LL = 6355 /UG_GENE = SCYA8




/UG_TITLE = small inducible cytokine subfamily A (Cys-Cys), member 8




(monocyte chemotacti


209583_s_at
AF063591
gb: AF063591.1 /DB_XREF = gi: 12002013 /FEA = FLmRNA /CNT = 108
chr3q12-q13
Hs.79015
CD200 antigen
CD200




/TID = Hs.79015.0 /TIER = FL + Stack /STK = 37 /UG = Hs.79015 /LL = 4345




/UG_GENE = MOX2 /DEF = Homo sapiens brain my033 protein mRNA,




complete cds. /PROD = brain my033 protein /FL = gb: AF063591.1


207277_at
AF290886
gb: AF290886.1 /DB_XREF = gi: 13383467 /FEA = FLmRNA /CNT = 8
chr19p13
Hs.278694
CD209 antigen
CD209




/TID = Hs.278694.0 /TIER = FL /STK = 0 /UG = Hs.278694 /LL = 30835




/UG_GENE = CD209 /DEF = Homo sapiens DC-SIGN mRNA, complete cds.




/PROD = DC-SIGN /FL = gb: AF290886.1 gb: M98457.1 gb: NM_021155.1


203416_at
NM_000560
gb: NM_000560.1 /DB_XREF = gi: 10834971 /GEN = CD53 /FEA = FLmRNA
chr1p13
Hs.443057
CD53 antigen
CD53




/CNT = 137 /TID = Hs.82212.0 /TIER = FL + Stack /STK = 64 /UG = Hs.82212




/LL = 963 /DEF = Homo sapiens CD53 antigen (CD53), mRNA. /PROD = CD53




antigen /FL = gb: NM_000560.1 gb: M60871.1 gb: M37033.1


218451_at
NM_022842
gb: NM_022842.1 /DB_XREF = gi: 12383093 /GEN = FLJ22969
chr3p21.31
Hs.146170
CUB domain containing protein 1
CDCP1




/FEA = FLmRNA /CNT = 97 /TID = Hs.146170.0 /TIER = FL + Stack /STK = 16




/UG = Hs.146170 /LL = 64866 /DEF = Homo sapiens hypothetical protein




FLJ22969 (FLJ22969), mRNA. /PROD = hypothetical protein FLJ22969




/FL = gb: NM_022842.1


217428_s_at
X98568
gb: X98568 /DB_XREF = gi: 1405722 /FEA = DNA /CNT = 2 /TID = Hs.179729.1
chr6q21-q22
Hs.179729
collagen, type X, alpha 1(Schmid
COL10A1




/TIER = ConsEnd /STK = 0 /UG = Hs.179729 /LL = 1300 /UG_GENE = COL10A1


metaphyseal chondrodysplasia)




/UG_TITLE = collagen, type X, alpha 1 (Schmid metaphyseal




chondrodysplasia) /DEF = H. sapiens type X collagen gene


205159_at
AV756141
gb: AV756141 /DB_XREF = gi: 10913989 /DB_XREF = AV756141
chr22q13.1
Hs.285401
colony stimulating factor 2
CSF2RB




/CLONE = BMFAKF10 /FEA = FLmRNA /CNT = 51 /TID = Hs.285401.0


receptor, beta, low-affinity




/TIER = Stack /STK = 20 /UG = Hs.285401 /LL = 1439 /UG_GENE = CSF2RB


(granulocyte-macrophage) ///




/UG_TITLE = colony stimulating factor 2 receptor, beta, low-affinity


colony stimulating factor 2 receptor,




(granulocyte-macrophage)


beta, low-affinity (granulocyte-







macrophage)


201487_at
NM_001814
gb: NM_001814.1 /DB_XREF = gi: 4503140 /GEN = CTSC /FEA = FLmRNA
chr11q14.1-q14.3
Hs.128065
cathepsin C
CTSC




/CNT = 257 /TID = Hs.10029.0 /TIER = FL + Stack /STK = 125 /UG = Hs.10029




/LL = 1075 /DEF = Homo sapiens cathepsin C (CTSC), mRNA.




/PROD = cathepsin C /FL = gb: NM_001814.1


203666_at
NM_000609
gb: NM_000609.1 /DB_XREF = gi: 10834987 /GEN = SDF1 /FEA = FLmRNA
chr10q11.1
Hs.436042
chemokine (C-X-C motif) ligand
CXCL12




/CNT = 114 /TID = Hs.237356.0 /TIER = FL + Stack /STK = 60 /UG = Hs.237356


12 (stromal cell-derived factor 1)




/LL = 6387 /DEF = Homo sapiens stromal cell-derived factor 1 (SDF1), mRNA.




/PROD = stromal cell-derived factor 1 /FL = gb: L36033.1 gb: NM_00060


223553_s_at
BC004564
gb: BC004564.1 /DB_XREF = gi: 13528734 /FEA = FLmRNA /CNT = 30
chr5q35.3
Hs.122559
docking protein 3
DOK3




/TID = Hs.122559.1 /TIER = FL + Stack /STK = 12 /UG = Hs.122559 /DEF = Homo




sapiens, Similar to hypothetical protein FLJ22570, clone MGC: 10476, mRNA,




complete cds. /PROD = Similar to hypothetical protein FLJ22570 /


219454_at
NM_015507
gb: NM_015507.2 /DB_XREF = gi: 13124887 /GEN = EGFL6 /FEA = FLmRNA
chrxp22
Hs.12844
EGF-like-domain, multiple 6
EGFL6




/CNT = 163 /TID = Hs.12844.0 /TIER = FL + Stack /STK = 40 /UG = Hs.12844




/LL = 25975 /DEF = Homo sapiens EGF-like-domain, multiple 6 (EGFL6),




mRNA. /PROD = epidermal growth factor-like protein 6precursor /FL = gb: NM—


1554899_s_at
BC020763
gb: BC020763.1 /DB_XREF = gi: 18088866 /TID = Hs2.433300.2 /CNT = 3
chr1q23
Hs.433300
Fc fragment of IgE, high affinity I,
FCER1G




/FEA = FLmRNA /TIER = FL /STK = 1 /LL = 2207 /UG_GENE = FCER1G


receptor for; gamma polypeptide




/UG = Hs.433300 /DEF = Homo sapiens, Similar to Fc fragment of IgE, high




affinity I, receptor for; gamma polypeptide, clone MGC: 22620 IMAGE: 470442


203561_at
NM_021642
gb: NM_021642.1 /DB_XREF = gi: 11056051 /GEN = FCGR2A
chr1q23
Hs.352642
Fc fragment of IgG, low affinity
FCGR2A




/FEA = FLmRNA /CNT = 142 /TID = Hs.78864.0 /TIER = FL + Stack /STK = 8


IIa, receptor (CD32)




/UG = Hs.78864 /LL = 2212 /DEF = Homo sapiens Fc fragment of IgG, low




affinity IIa, receptor for (CD32) (FCGR2A), mRNA. /PROD = Fc fragment of




IgG, low affi


218468_s_at
AF154054
gb: AF154054.1 /DB_XREF = gi: 10863087 /GEN = DRM /FEA = FLmRNA
chr15q13-q15
Hs.40098
gremlin 1, cysteine knot
GREM1




/CNT = 228 /TID = Hs.40098.0 /TIER = FL + Stack /STK = 20 /UG = Hs.40098


superfamily, homolog (Xenopus




/LL = 26585 /DEF = Homo sapiens DRM (DRM) mRNA, complete cds.


laevis)




/PROD = DRM /FL = gb: NM_013372.1 gb: AF110137.2 gb: AF045800.1




gb: AF154054.1


217478_s_at
X76775
gb: X76775 /DB_XREF = gi: 512468 /FEA = DNA_1 /CNT = 1 /TID = Hs.77522.1
chr6p21.3
Hs.351279
major histocompatibility complex,
HLA-DMA




/TIER = ConsEnd /STK = 0 /UG = Hs.77522 /LL = 3108 /UG_GENE = HLA-DMA


class II, DM alpha




/UG_TITLE = major histocompatibility complex, class II, DM alpha




/DEF = H. sapiens HLA-DMA gene


203932_at
NM_002118
gb: NM_002118.1 /DB_XREF = gi: 4504398 /GEN = HLA-DMB
chr6p21.3
Hs.1162
major histocompatibility complex,
HLA-DMB




/FEA = FLmRNA /CNT = 91 /TID = Hs.1162.0 /TIER = FL + Stack /STK = 49


class II, DM beta /// major




/UG = Hs.1162 /LL = 3109 /DEF = Homo sapiens major histocompatibility


histocompatibility complex, class II,




complex, class II, DM beta (HLA-DMB), mRNA. /PROD = major


DM beta




histocompatibility complex, c


211991_s_at
M27487
gb: M27487.1 /DB_XREF = gi: 703088 /GEN = HLA-DPA1 /FEA = FLmRNA
chr6p21.3
Hs.914
major histocompatibility complex,
HLA-DPA1




/CNT = 358 /TID = Hs.914.0 /TIER = FL + Stack /STK = 139 /UG = Hs.914


class II, DP alpha 1




/DEF = Homo sapiens MHC class II DPw3-alpha-1 chain mRNA, complete




cds. /PROD = MHC class II DP3-alpha /FL = gb: M27487.1


212671_s_at
BG397856
gb: BG397856 /DB_XREF = gi: 13291304 /DB_XREF = 602438950F1
chr6p21.3
Hs.387679
major histocompatibility complex,
HLA-DQA1 ///




/CLONE = IMAGE: 4564956 /FEA = mRNA /CNT = 167 /TID = Hs.198253.2


class II, DQ alpha 1 /// major
HLA-DQA2




/TIER = Stack /STK = 59 /UG = Hs.198253 /LL = 3117 /UG_GENE = HLA-DQA1


histocompatibility complex, class II,




/UG_TITLE = major histocompatibility complex, class II, DQ alpha 1


DQ alpha 2


208894_at
M60334
gb: M60334.1 /DB_XREF = gi: 188255 /GEN = HLA-DRA /FEA = FLmRNA
chr6p21.3
Hs.409805
major histocompatibility complex,
HLA-DRA




/CNT-470 /TID = Hs.76807.0 /TIER = FL /STK = 0 /UG = Hs.76807 /LL = 3122


class II, DR alpha /// major




/DEF = Human MHC class II HLA-DR-alpha mRNA, complete cds.


histocompatibility complex, class II,




/PROD = cell surface glycoprotein /FL = gb: M60334.1 gb: NM_019111.1


DR alpha


204670_x_at
NM_002125
gb: NM_002125.1 /DB_XREF = gi: 4504412 /GEN = HLA-DRB5
chr6p21.3
Hs.308026
major histocompatibility complex,
HLA-DRB1




/FEA = FLmRNA /CNT = 62 /TID = Hs.308026.0 /TIER = FL /STK = 1


class II, DR beta 1




/UG = Hs.308026 /LL = 3127 /DEF = Homo sapiens major histocompatibility




complex, class II, DR beta 5 (HLA-DRB5), mRNA. /PROD = major




histocompatibility complex,


209312_x_at
U65585
gb: U65585.1 /DB_XREF = gi: 5478215 /GEN = HLA-DRB1 /FEA = FLmRNA


major histocompatibility complex,
HLA-DRB1




/CNT = 126 /TID = Hs.180255.0 /TIER = FL /STK = 0 /UG = Hs.180255 /LL = 3123


class II, DR beta /// major




/DEF = Homo sapiens MHC class II antigen (HLA-DRB1) mRNA, HLA-


histocompatibility complex, class II,




DRB1*PBL allele, complete cds. /PROD = MHC class II antigen


DR beta 1




/FL = gb: NM_0021


208306_x_at
NM_021983
gb: NM_021983.2 /DB_XREF = gi: 11875206 /GEN = HLA-DRB4
chr6p21.3
Hs.308026
Major histocompatibility complex,
HLA-DRB3




/FEA = FLmRNA /CNT = 2 /TID = Hs.293934.0 /TIER = FL /STK = 0


class II, DR beta 3




/UG = Hs.293934 /LL = 3126 /DEF = Homo sapiens major histocompatibility




complex, class II, DR beta 4 (HLA-DRB4), mRNA. /PROD = major




histocompatibility complex,


217362_x_at
AF005487
gb: AF005487.1 /DB_XREF = gi: 5915893 /FEA = mRNA /CNT = 2
chr6p21.3

major histocompatibility complex,
HLA-DRB6




/TID = Hs.167385.0 /TIER = ConsEnd /STK = 0 /UG = Hs.167385


class II, DR beta 6 (pseudogene)




/UG_TITLE = Homo sapiens MHC class II antigen HLA-DRB6 mRNA, partial




cds /DEF = Homo sapiens MHC class II antigen (DRB6) mRNA, HLA-




DRB6*0201 allele, sequenc


229400_at
AW299531
gb: AW299531 /DB_XREF = gi: 6709208 /DB_XREF = xs51a03.x1
chr2q31.1
Hs.123070
homeo box D10
HOXD10




/CLONE = IMAGE: 2773132 /FEA = EST /CNT = 17 /TID = Hs.188023.0




/TIER = Stack /STK = 10 /UG = Hs.188023 /UG_TITLE = ESTs


209374_s_at
BC001872
gb: BC001872.1 /DB_XREF = gi: 12804852 /FEA = FLmRNA /CNT = 302
chr14q32.33
Hs.439852
immunoglobulin heavy constant
IGHM




/TID = Hs.302063.0 /TIER = FL + Stack /STK = 207 /UG = Hs.302063 /LL = 3507


mu




/UG_GENE = IGHM /DEF = Homo sapiens, clone MGC: 1228, mRNA, complete




cds. /PROD = Unknown (protein for MGC: 1228) /FL = gb: BC002963.1




gb: BC001872


214669_x_at
BG485135
gb: BG485135 /DB_XREF = gi: 13417414 /DB_XREF = 602503756F1
chr2p12
Hs.377975 ///
Immunoglobulin kappa variable
IGKC




/CLONE = IMAGE: 4617445 /FEA = mRNA /CNT = 101 /TID = Hs.325722.1

Hs.449606 ///
1-5




/TIER = ConsEnd /STK = 0 /UG = Hs.325722 /LL = 28875 /UG_GENE = IGKV3D-

Hs.494060 ///




15 /UG_TITLE = immunoglobulin kappa variable 3D-15

Hs.512126 ///






Hs.534005


226218_at
BE217880
gb: BE217880 /DB = XREF = gi: 8905198 /DB_XREF = hv31a11.x1
chr5p13
Hs.362807
Interleukin 7 receptor
IL7R




/CLONE = IMAGE: 3175004 /FEA = EST /CNT = 52 /TID = Hs.237868.1




/TIER = Stack /STK = 24 /UG = Hs.237868 /LL = 3575 /UG_GENE = IL7R




/UG_TITLE = interleukin 7 receptor


205786_s_at
NM_000632
gb: NM_000632.2 /DB_XREF = gi: 6006013 /GEN = ITGAM /FEA = FLmRNA
chr16p11.2
Hs.172631
integrin, alpha M (complement
ITGAM




/CNT = 40 /TID = Hs.172631.0 /TIER = FL /STK = 1 /UG = Hs.172631 /LL = 3684


component receptor 3, alpha; also




/DEF = Homo sapiens integrin, alpha M (complement component receptor 3,


known as CD11b (p170),




alpha; also known as CD11b (p170), macrophage antigen alpha pol


macrophage antigen alpha







polypeptide) /// integrin, alpha M







(complement component receptor







3, alpha; also known as CD11b







(p170), macrophage antigen alpha







polypeptide)


202803_s_at
NM_000211
gb: NM_000211.1 /DB_XREF = gi: 4557885 /GEN = ITGB2 /FEA = FLmRNA
chr21q22.3
Hs.375957
integrin, beta 2 (antigen CD18
ITGB2




/CNT = 176 /TID = Hs.83968.0 /TIER = FL + Stack /STK = 80 /UG = Hs.83968


(p95), lymphocyte function-




/LL = 3689 /DEF = Homo sapiens integrin, beta 2 (antigen CD18 (p95),


associated antigen 1; macrophage




lymphocyte function-associated antigen 1; macrophage antigen 1 (mac-1)


antigen 1 (mac-1) beta subunit)


228167_at
AW574798
gb: AW574798 /DB_XREF = gi: 7246337 /DB_XREF = UI-HF-BK0-abh-e-04-0-
chr3q27.3
Hs.439354
kelch-like 6 (Drosophila)
KLHL6




UI.s1 /CLONE = IMAGE: 3056335 /FEA = mRNA /CNT = 35 /TID = Hs.43616.0




/TIER = Stack /STK = 18 /UG = Hs.43616 /UG_TITLE = Homo sapiens mRNA for




FLJ00029 protein, partial cds


205306_x_at
AI074145
gb: AI074145 /DB_XREF = gi: 3400789 /DB_XREF = ov13a06.x1
chr1q42-q44
Hs.409081
kynurenine 3-monooxygenase
KMO




/CLONE = IMAGE: 1637170 /FEA = FLmRNA /CNT = 48 /TID = Hs.107318.0


(kynurenine 3-hydroxylase)




/TIER = Stack /STK = 10 /UG = Hs.107318 /LL = 8564 /UG_GENE = KMO




/UG_TITLE = kynurenine 3-monooxygenase (kynurenine 3-hydroxylase)




/FL = gb: NM_003679.1 gb: AF0


201720_s_at
AI589086
gb: AI589086 /DB_XREF = gi: 4598134 /DB_XREF = tf80g10.x1
chr1p34
Hs.436200
lysosomal associated
LAPTM5




/CLONE = IMAGE: 2105634 /FEA = FLmRNA /CNT = 274 /TID = Hs.79356.0


multispanning membrane protein 5




/TIER = Stack /STK = 22 /UG = Hs.79356 /LL = 7805 /UG_GENE = LAPTM5




/UG_TITLE = Lysosomal-associated multispanning membrane protein-5




/FL = gb: NM_006762.1 gb: U


206140_at
NM_004789
gb: NM_004789.1 /DB_XREF = gi: 4758673 /GEN = LHX2 /FEA = FLmRNA
chr9q33-q34.1
Hs.1569
LIM homeobox 2
LHX2




/CNT = 18 /TID = Hs.1569.0 /TIER = FL /STK = 1 /UG = Hs.1569 /LL = 9355




/DEF = Homo sapiens LIM homeobox protein 2 (LHX2), mRNA. /PROD = LIM




homeobox protein 2 /FL = gb: NM_004789.1 gb: AF124735.1


213909_at
AU147799
gb: AU147799 /DB_XREF = gi: 11009320 /DB_XREF = AU147799
chr3q29
Hs.288467
leucine rich repeat containing 15
LRRC15




/CLONE = MAMMA1001744 /FEA = mRNA /CNT = 43 /TID = Hs.288467.0




/TIER = Stack /STK = 20 /UG = Hs.288467 /UG_TITLE = Homo sapiens cDNA




FLJ12280 fis, clone MAMMA1001744


213975_s_at
AV711904
gb: AV711904 /DB_XREF = gi: 10731210 /DB_XREF = AV711904
chr19q13.4 ///
Hs.149924
lysozyme (renal amyloidosis) ///
LYZ /// LILRB1




/CLONE = DCAAIE08 /FEA = mRNA /CNT = 21 /TID = Hs.277431.0 /TIER = Stack
chr12q15

leukocyte immunoglobulin-like




/STK = 8 /UG = Hs.277431 /UG_TITLE = Homo sapiens cDNA: FLJ23356 fis,


receptor, subfamily B (with TM and




clone HEP14919


ITIM domains), member 1


203435_s_at
NM_007287
gb: NM_007287.1 /DB_XREF = gi: 6042199 /GEN = MME /FEA = FLmRNA
chr3q25.1-q25.2
Hs.307734
membrane metallo-
MME




/CNT = 219 /TID = Hs.1298.2 /TIER = FL + Stack /STK = 59 /UG = Hs.1298


endopeptidase (neutral




/LL = 4311 /DEF = Homo sapiens membrane metallo-endopeptidase (neutral


endopeptidase, enkephalinase,




endopeptidase, enkephalinase, CALLA, CD10) (MME), transcript variant 1bis


CALLA, CD10)


204580_at
NM_002426
gb: NM_002426.1 /DB_XREF = gi: 4505206 /GEN = MMP12 /FEA = FLmRNA
chr11q22.3
Hs.1695
matrix metallopeptidase 12
MMP12




/CNT = 72 /TID = Hs.1695.0 /TIER = FL + Stack /STK = 18 /UG = Hs.1695 /LL = 4321


(macrophage elastase)




/DEF = Homo sapiens matrix metalloproteinase 12 (macrophage elastase)




(MMP12), mRNA. /PROD = matrix metalloproteinase 12 preproprotein


226844_at
AI375115
gb: AI375115 /DB_XREF = gi: 4175105 /DB_XREF = tc09e10.x1
chr9p21.2
Hs.128905
MOB1, Mps One Binder kinase
MOBKL2B




/CLONE = IMAGE: 2063370 /FEA = EST /CNT = 43 /TID = Hs.293849.0


activator-like 2B (yeast)




/TIER = Stack /STK = 27 /UG = Hs.293849 /UG_TITLE = ESTs


226818_at
T64884
gb: T64884 /DB_XREF = gi: 673929 /DB_XREF = yd10b06.s1
chr11q12.1
Hs.62264
macrophage expressed gene 1
MPEG1




/CLONE = IMAGE: 66707 /FEA = mRNA /CNT = 72 /TID = Hs.288581.0




/TIER = Stack /STK = 8 /UG = Hs.288581 /UG_TITLE = Homo sapiens cDNA




FLJ14296 fis, clone PLACE1008455


204438_at
NM_002438
gb: NM_002438.1 /DB_XREF = gi: 4505244 /GEN = MRC1 /FEA = FLmRNA
chr10p13
Hs.75182
mannose receptor, C type 1 ///
MRC1 ///




/CNT = 64 /TID = Hs.75182.0 /TIER = FL + Stack /STK = 25 /UG = Hs.75182


mannose receptor. C type 1-like 1
MRC1L1




/LL = 4360 /DEF = Homo sapiens mannose receptor, C type 1 (MRC1), mRNA.




/PROD = mannose receptor, C type 1 /FL = gb: NM_002438.1 gb: J05550.1


1555728_a_at
AF354928
gb: AF354928.1 /DB_XREF = gi: 15808758 /TID = Hs2.325960.4 /CNT = 1
chr11q12
Hs.325960
membrane-spanning 4-domains,
MS4A4A




/FEA = FLmRNA /TIER = FL /STK = 1 /LL = 51338 /UG_GENE = MS4A4A


subfamily A, member 4




/UG = Hs.325960 /DEF = Homo sapiens MS4A4A protein mRNA, complete cds,




alternatively spliced. /PROD = MS4A4A protein /FL = gb: AF354928.1


209949_at
BC001606
gb: BC001606.1 /DB_XREF = gi: 12804408 /FEA = FLmRNA /CNT = 31
chr1q25
Hs.949
neutrophil cytosolic factor 2
NCF2




/TID = Hs.949.0 /TIER = FL /STK = 4 /UG = Hs.949 /LL = 4688 /UG_GENE = NCF2


(65 kDa, chronic granulomatous




/DEF = Homo sapiens, Similar to neutrophil cytosolic factor 2 (65 kD, chronic


disease, autosomal 2)




granulomatous disease, autosomal 2), clone MGC: 2275, mRNA, co


221210_s_at
NM_030769
gb: NM_030769.1 /DB_XREF = gi: 13540532 /GEN = C1ORF13
chr1q25
Hs.64896
N-acetylneuraminate pyruvate
NPL




/FEA = FLmRNA /CNT = 1 /TID = HsAffx.900046.172 /TIER = FL /STK = 0


lyase (dihydrodipicolinate




/DEF = Homo sapiens hypothetical protein similar to swine acylneuraminate


synthase) /// N-acetylneuraminate




lyase (C1ORF13), mRNA. /PROD = hypothetical protein similar to


pyruvate lyase (dihydrodipicolinate




swineacylneuram


synthase)


218625_at
NM_016588
gb: NM_016588.1 /DB_XREF = gi: 7706122 /GEN = LOC51299
chr6p25.1
Hs.103291
neuritin 1
NRN1




/FEA = FLmRNA /CNT = 106 /TID = Hs.103291.0 /TIER = FL + Stack /STK = 36




/UG = Hs.103291 /LL = 51299 /DEF = Homo sapiens neuritin (LOC51299),




mRNA. /PROD = neuritin /FL = gb: NM_016588.1 gb: BC002683.1




gb: AF136631.1


231867_at
AB032953
gb: AB032953.1 /DB_XREF = gi: 6329762 /GEN = KIAA1127 /FEA = mRNA
chr5q34
Hs.173560
odz, odd Oz/ten-m homolog 2
ODZ2




/CNT = 32 /TID = Hs.173560.0 /TIER = ConsEnd /STK = 2 /UG = Hs.173560


(Drosophila)




/LL = 57451 /DEF = Homo sapiens mRNA for KIAA1127 protein, partial cds.




/PROD = KIAA1127 protein


226459_at
AW575754
gb: AW575754 /DB_XREF = gi: 7247293 /DB_XREF = UI-HF-BM0-adw-c-06-
chr10q24.1
Hs.374836
phosphoinositide-3-kinase
PIK3AP1




0-UI.s1 /CLONE = IMAGE: 3063154 /FEA = EST /CNT = 60 /TID = Hs.86437.0


adaptor protein 1




/TIER = Stack /STK = 35 /UG = Hs.86437 /UG_TITLE = ESTs, Highly similar to




AF219140 1 gastric cancer-related protein GCYS-20 (H. sapiens)


206214_at
NM_005084
gb: NM_005084.1 /DB_XREF = gi: 4826883 /GEN = PLA2G7 /FEA = FLmRNA
chr6p21.2-p12
Hs.93304
phospholipase A2, group VII
PLA2G7




/CNT = 21 /TID = Hs.93304.0 /TIER = FL + Stack /STK = 8 /UG = Hs.93304


(platelet-activating factor




/LL = 7941 /DEF = Homo sapiens phospholipase A2, group VII (platelet-


acetylhydrolase, plasma) ///




activating factor acetylhydrolase, plasma) (PLA2G7), mRNA.


phospholipase A2, group VII




/PROD = phospho


(platelet-activating factor







acetylhydrolase, plasma)


212588_at
Y00062
gb: Y00062.1 /DB_XREF = gi: 34275 /FEA = mRNA /CNT = 145
chr1q31-q32
Hs.444324
protein tyrosine phosphatase,
PTPRC




/TID = Hs.170121.1 /TIER = Stack /STK = 18 /UG = Hs.170121 /LL = 5788


receptor type, C




/UG_GENE = PTPRC /UG_TITLE = protein tyrosine phosphatase, receptor




type, C /DEF = Human mRNA for T200 leukocyte common antigen (CD45,




LC-A).


37793_r_at
AF034956
Cluster Incl. AF034956: Homo sapiens RAD51D mRNA, complete cds
chr17q11
Hs.125244
RAD51-like 3 (S. cerevisiae)
RAD51L3




/cds = (124,993) /gb = AF034956 /gi = 2920581 /ug = Hs.125244 /len = 1564


203185_at
NM_014737
gb: NM_014737.1 /DB_XREF = gi: 7661963 /GEN = RASSF2 /FEA = FLmRNA
chr20pter-p12.1
Hs.80905
Ras association (RaIGDS/AF-6)
RASSF2




/CNT = 152 /TID = Hs.80905.0 /TIER = FL + Stack /STK = 30 /UG = Hs.80905


domain family 2




/LL = 9770 /DEF = Homo sapiens Ras association (RalGDSAF-6) domain




family 2 (RASSF2), mRNA. /PROD = Ras association (RalGDSAF-6) domain




famil


225763_at
AI659418
gb: AI659418 /DB_XREF = gi: 4762988 /DB_XREF = tu30a07.x1
chr1q22-q24
Hs.233125
RCSD domain containing 1
RCSD1




/CLONE = IMAGE: 2252532 /FEA = mRNA /CNT = 77 /TID = Hs.14040.0




/TIER = Stack /STK = 23 /UG = Hs.14040 /UG_TITLE = Homo sapiens cDNA:




FLJ21772 fis, clone COLF7808


213566_at
NM_005615
gb: NM_005615.1 /DB_XREF = gi: 5032044 /GEN = RNASE6 /FEA = FLmRNA
chr14q11.2
Hs.23262
ribonuclease, RNase A family, k6
RNASE6




/CNT = 53 /TID = Hs.23262.0 /TIER = FL + Stack /STK = 30 /UG = Hs.23262


/// ribonuclease, RNase A family,




/LL = 6039 /DEF = Homo sapiens ribonuclease, RNase A family, k6 (RNASE6),


k6




mRNA. /PROD = ribonuclease, RNase A family, k6 /FL = gb: NM_005615.1


219385_at
NM_020125
gb: NM_020125.1 /DB_XREF = gi: 9910341 /GEN = SBBI42 /FEA = FLmRNA
chr1q23.2
Hs.438683
SLAM family member 8
SLAMF8




/CNT = 37 /TID = Hs.20450.0 /TIER = FL /STK = 0 /UG = Hs.20450 /LL = 56833




/DEF = Homo sapiens BCM-like membrane protein precursor (SBBI42),




mRNA. /PROD = BCM-like membrane protein precursor /FL = gb: NM_020125.1




gb


203473_at
NM_007256
gb: NM_007256.1 /DB_XREF = gi: 6005819 /GEN = SLC21A9 /FEA = FLmRNA
chr11q13
Hs.7884
solute carrier organic anion
SLCO2B1




/CNT = 120 /TID = Hs.7884.0 /TIER = FL + Stack /STK = 25 /UG = Hs.7884


transporter family, member 2B1




/LL = 11309 /DEF = Homo sapiens solute carrier family 21 (organic anion




transporter), member 9 (SLC21A9), mRNA. /PROD = solute carrier family


240715_at
AW269421
gb: AW269421 /DB_XREF = gi: 6656451 /DB_XREF = xv42e03.x1
chr12q24.1
Hs.381715
T-box 5
TBX5




/CLONE = IMAGE: 2815804 /FEA = EST /CNT = 4 /TID = Hs.128093.0




/TIER = ConsEnd /STK = 4 /UG = Hs.128093 /UG_TITLE = ESTs


205844_at
NM_004666
gb: NM_004666.1 /DB_XREF = gi: 4759311 /GEN = VNN1 /FEA = FLmRNA
chr6q23-q24
Hs.12114
vanin 1 /// vanin 1
VNN1




/CNT = 36 /TID = Hs.12114.0 /TIER = FL + Stack /STK = 13 /UG = Hs.12114




/LL = 8876 /DEF = Homo sapiens vanin 1 (VNN1), mRNA. /PROD = vanin 1




/FL = gb: U39664.1 gb: NM_004666.1


205922_at
NM_004665
gb: NM_004665.1 /DB_XREF = gi: 4759313 /GEN = VNN2 /FEA = FLmRNA


vanin 2 /// vanin 2
VNN2




/CNT = 24 /TID = Hs.121102.0 /TIER = FL + Stack /STK = 13 /UG = Hs.121102




/LL = 8875 /DEF = Homo sapiens vanin 2 (VNN2), mRNA. /PROD = vanin 2




/FL = gb: D89974.1 gb: NM_004665.1


227346_at
AI741188
gb: AI741188 /DB_XREF = gi: 5109476 /DB_XREF = wg26a11.x1


Zinc finger protein, subfamily 1A,
ZNFN1A1




/CLONE = IMAGE: 2366204 /FEA = EST /CNT = 36 /TID = Hs.121587.0


1 (Ikaros)




/TIER = Stack /STK = 12 /UG = Hs.121587 /UG_TITLE = ESTs


214836_x_at
BG536224
gb: BG536224 /DB_XREF = gi: 13527769 /DB_XREF = 602565445F1

Hs.469271 ///
Immunoglobulin kappa light
—




/CLONE = IMAGE: 4690258 /FEA = DNA /CNT = 36 /TID = Hs.123030.0

Hs.525895 ///
chain VJ region (ID POM010) ///




/TIER = ConsEnd /STK = 0 /UG = Hs.123030 /UG_TITLE = Human kappa-

Hs.534006
Immunoglobulin kappa light chain




immunoglobulin germline pseudogene (Chr22.4) variable region (subgroup V


VJ region (ID POM022) /// (clone




kappa II)


TR1.6VL) anti-thyroid peroxidase







monoclonal autoantibody IgK







chain, V region


219947_at
NM_016184
gb: NM_016184.1 /DB_XREF = gi: 7705337 /GEN = CLECSF6
chr12p13
Hs.115515
C-type (calcium dependent,
CLECSF6




/FEA = FLmRNA /CNT = 15 /TID = Hs.115515.0 /TIER = FL /STK = 7


carbohydrate-recognition domain)




/UG = Hs.115515 /LL = 50856 /DEF = Homo sapiens C-type (calcium


lectin, superfamily member 6




dependent, carbohydrate-recognition domain) lectin, superfamily member 6




(CLECSF6), mRNA. /PROD=







endothelial differentiation,
EDG3







sphingolipid G-protein-coupled







receptor, 3







immunoglobulin heavy constant
IGHA1; IGHA2;







alpha 1 /// immunoglobulin heavy
MGC27165







constant alpha 2 (A2m marker) ///







hypothetical protein MGC27165







mast cell-expressed membrane
MCEMP1







protein 1







protein kinase substrate MK2S4
MK2S4






















TABLE 2





Probe set
Public ID

Chromosomal





ID number
number (NCBI)
Target description
location
Unigene ID
Gene Title
Gene Symbol







206046_at
NM_003812
gb: NM_003812.1 /DB_XREF = gi: 4501912 /GEN = ADAM23 /FEA = FLmRNA
chr2q33
Hs.432317
ADAM metallopeptidase domain 23
ADAM23




/CNT = 26 /TID = Hs.7164.0 /TIER = FL + Stack /STK = 11 /UG = Hs.7164 /LL = 8745




/DEF = Homo sapiens a disintegrin and metalloproteinase domain 23




(ADAM23), mRNA. /PROD = a disintegrin and metalloproteinase domain


206170_at
NM_000024
gb: NM_000024.2 /DB_XREF = gi: 13162366 /GEN = ADRB2 /FEA = FLmRNA


adrenergic, beta-2-, receptor,
ADRB2




/CNT = 35 /TID = Hs.2551.0 /TIER = FL /STK = 0 /UG = Hs.2551 /LL = 154


surface




/DEF = Homo sapiens adrenergic, beta-2-, receptor, surface (ADRB2), mRNA.




/PROD = adrenergic, beta-2-, receptor, surface /FL = gb: NM_000024.2 g


205357_s_at
NM_000685
gb: NM_000685.2 /DB_XREF = gi: 6715581 /GEN = AGTR1 /FEA = FLmRNA
chr3q21-q25
Hs.89472
angiotensin II receptor, type 1
AGTR1




/CNT = 61 /TID = Hs.89472.0 /TIER = FL + Stack /STK = 18 /UG = Hs.89472




/LL = 185 /DEF = Homo sapiens angiotensin receptor 1 (AGTR1), transcript




variant 1, mRNA. /PROD = angiotensin receptor 1 /FL = gb: M93394.1 gb: NM—


216594_x_at
S68290
gb: S68290.1 /DB_XREF = gi: 544763 /GEN = chlordecone reductase homolog
chr10p15-p14
Hs.201967
aldo-keto reductase family 1,
AKR1C1




/FEA = mRNA /CNT = 1 /TID = Hs.306098.1 /TIER = ConsEnd /STK = 0


member C1 (dihydrodiol




/UG = Hs.306098 /LL = 1645 /UG_TITLE = aldo-keto reductase family 1, member


dehydrogenase 1; 20-alpha (3-




C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxystero


alpha)-hydroxysteroid







dehydrogenase)


209699_x_at
U05598
gb: U05598.1 /DB_XREF = gi: 531159 /FEA = FLmRNA /CNT = 124
chr10p15-p14
Hs.201967
aldo-keto reductase family 1,
AKR1C2




/TID = Hs.201967.0 /TIER = FL + Stack /STK = 29 /UG = Hs.201967 /LL = 1646


member C2 (dihydrodiol




/UG_GENE = AKR1C2 /DEF = Human dihydrodiol dehydrogenase mRNA,


dehydrogenase 2; bile acid binding




complete cds. /PROD = dihydrodiol dehydrogenase /FL = gb: U05598.1


protein; 3-alpha hydroxysteroid




gb: AB031083.1 gb: AB


dehydrogenase, type III)


223093_at
T99215
gb: T99215 /DB_XREF = gi: 748952 /DB_XREF = ye63a06.s1
chr5p15.1
Hs.156727
ankylosis, progressive homolog
ANKH




/CLONE = IMAGE: 122386 /FEA = FLmRNA /CNT = 174 /TID = Hs.168640.1


(mouse)




/TIER = Stack /STK = 13 /UG = Hs.168640 /LL = 56172 /UG_GENE = ANKH




/UG_TITLE = ankylosis, progressive (mouse) homolog /FL = gb: AF274753.1


227827_at
AW138143
gb: AW138143 /DB_XREF = gi: 6142543 /DB_XREF = UI-H-BI1-acy-b-09-0-


Sorbin and SH3 domain containing 2
ARGBP2




UI.s1 /CLONE = IMAGE: 2715976 /FEA = EST /CNT = 32 /TID = Hs.71721.0




/TIER = Stack /STK = 16 /UG = Hs.71721 /UG_TITLE = ESTs


230309_at
BE876610
gb: BE876610/DB_XREF = gi: 10325386 /DB_XREF = 601487767F1


Betaine-homocysteine
BHMT2




/CLONE = IMAGE: 3889936 /FEA = EST /CNT = 13 /TID = Hs.172382.1


methyltransferase 2




/TIER = Stack /STK = 11 /UG = Hs.172382 /LL = 23743 /UG_GENE = BHMT2




/UG_TITLE = betaine-homocysteine methyltransferase 2


206176_at
NM_001718
gb: NM_001718.2 /DB_XREF = gi: 4809281 /GEN = BMP6 /FEA = FLmRNA
chr6p24-p23
Hs.285671
bone morphogenetic protein 6
BMP6




/CNT = 18 /TID = Hs.285671.0 /TIER = FL /STK = 0 /UG = Hs.285671 /LL = 654




/DEF = Homo sapiens bone morphogenetic protein 6 (BMP6), mRNA.




/PROD = bone morphogenetic protein 6 precursor /FL = gb: M60315.1




gb: NM_001718.


241412_at
AI620677
gb: AI620677 /DB_XREF = gi: 4629803 /DB_XREF = tu85e09.x1
chr4q13-q21
Hs.73105
betacellulin
BTC




/CLONE = IMAGE: 2257864 /FEA = EST /CNT = 7 /TID = Hs.154191.0




/TIER = ConsEnd /STK = 0 /UG = Hs.154191 /UG_TITLE = ESTs


224458_at
BC006115
gb: BC006115.1 /DB_XREF = gi: 13543948 /FEA = FLmRNA /CNT = 1
chr9q31.1
Hs.431270
chromosome 9 open reading frame
C9orf125




/TID = HsAffx.900859.640 /TIER = FL /STK = 0 /DEF = Homo sapiens, Similar to


125 /// chromosome 9 open reading




RIKEN cDNA 2810432L12 gene, clone MGC: 12992, mRNA, complete cds.


frame 125




/PROD = Similar to RIKEN cDNA 2810432L12 gene /FL = gb: BC006115.1


235182_at
AI816793
gb: AI816793 /DB_XREF = gi: 5435872 /DB_XREF = wj34b11.x1
chr20p12.1
Hs.156650
chromosome 20 open reading
C20orf82




/CLONE = IMAGE: 2404701 /FEA = EST /CNT = 13 /TID = Hs.135100.0


frame 82




/TIER = ConsEnd /STK = 4 /UG = Hs.135100 /UG_TITLE = ESTs


227226_at
AA418816
gb: AA418816 /DB_XREF = gi: 2080617 /DB_XREF = zw01a04.s1
chr6q14.3
Hs.425033
chromosome 6 open reading frame
C6orf117




/CLONE = IMAGE: 767982 /FEA = EST /CNT = 34 /TID = Hs.20953.0 /TIER = Stack


117




/STK = 24 /UG = Hs.20953 /UG_TITLE = ESTs


209616_s_at
S73751
gb: S73751.1 /DB_XREF = gi: 688112 /FEA = FLmRNA /CNT = 178
chr16q13-q22.1
Hs.278997
carboxylesterase 1
CES1




/TID = Hs.76688.0 /TIER = FL /STK = 0 /UG = Hs.76688 /LL = 1066


(monocyte/macrophage serine




/UG_GENE = CES1 /DEF = Homo sapiens acyl coenzyme A: cholesterol


esterase 1)




acyltransferase mRNA, complete cds. /PROD = acyl coenzyme A: cholesterol




acyltransferase /FL


208791_at
M25915
gb: M25915.1 /DB_XREF = gi: 180619 /FEA = FLmRNA /CNT = 470
chr8p21-p12
Hs.436657
clusterin (complement lysis
CLU




/TID = Hs.75106.0 /TIER = FL /STK = 3 /UG = Hs.75106 /LL = 1191


inhibitor, SP-40,40, sulfated




/UG_GENE = CLU /UG_TITLE = clusterin (complement lysis inhibitor, SP-40,40,


glycoprotein 2, testosterone-




sulfated glycoprotein 2, testosterone-repressed prostate message 2,


repressed prostate message 2,




apolipopr


apolipoprotein J)


205713_s_at
NM_000095
gb: NM_000095.1 /DB_XREF = gi: 4557482 /GEN = COMP /FEA = FLmRNA
chr19p13.1
Hs.1584
cartilage oligomeric matrix protein
COMP




/CNT = 64 /TID = Hs.1584.0 /TIER = FL + Stack /STK = 16 /UG = Hs.1584 /LL = 1311




/DEF = Homo sapiens cartilage oligomeric matrix protein




(pseudoachondroplasia, epiphyseal dysplasia 1, multiple) (COMP), mRNA.




/PROD =


206315_at
NM_004750
gb: NM_004750.1 /DB_XREF = gi: 4758061 /GEN = CRLF1 /FEA = FLmRNA
chr19p12
Hs.114948
cytokine receptor-like factor 1
CRLF1




/CNT = 20 /TID = Hs.114948.0 /TIER = FL + Stack /STK = 13 /UG = Hs.114948




/LL = 9244 /DEF = Homo sapiens cytokine receptor-like factor 1 (CRLF1),




mRNA. /PROD = cytokine receptor-like factor 1 /FL = gb: AF073515.1 gb: NM


208350_at
NM_001890
gb: NM_001890.1 /DB_XREF = gi: 4503084 /GEN = CSN1 /FEA = FLmRNA
chr4q21.1
Hs.3155
casein alpha s1
CSN1S1




/CNT = 4 /TID = Hs.3155.0 /TIER = FL /STK = 0 /UG = Hs.3155 /LL = 1446




/DEF = Homo sapiens casein, alpha (CSN1), mRNA. /PROD = casein, alpha




/FL = gb: NM_001890.1 gb: U23157.1


202435_s_at
AU154504
gb: AU154504 /DB_XREF = gi: 11016025 /DB_XREF = AU154504
chr2p21
Hs.154654
cytochrome P450, family 1,
CYP1B1




/CLONE = NT2RP4001328 /FEA = FLmRNA /CNT = 212 /TID = Hs.154654.0


subfamily B, polypeptide 1




/TIER = Stack /STK = 20 /UG = Hs.154654 /LL = 1545 /UG_GENE = CYP1B1




/UG_TITLE = cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1




(glaucoma 3, p


205818_at
NM_014618
gb: NM_014618.1/DB_XREF = gi: 7657008 /GEN = DBCCR1 /FEA = FLmRNA
chr9q32-q33
Hs.6090
deleted in bladder cancer 1
DBC1




/CNT = 33 /TID = Hs.6090.0 /TIER = FL + Stack /STK = 8 /UG = Hs.6090 /LL = 1620




/DEF = Homo sapiens deleted in bladder cancer chromosome region




candidate 1 (DBCCR1), mRNA. /PROD = deleted in bladder cancer chromoso


210397_at
U73945
gb: U73945.1 /DB_XREF = gi: 1755147 /FEA = FLmRNA /CNT = 26
chr8p23.2-p23.1
Hs.32949
defensin, beta 1
DEFB1




/TID = Hs.32949.0 /TIER = FL /STK = 0 /UG = Hs.32949 /LL = 1672




/UG_GENE = DEFB1 /DEF = Human beta-defensin-1 mRNA, complete cds.




/PROD = beta-defensin-1 /FL = gb: U73945.1 gb: NM_005218.2


205554_s_at
NM_004944
gb: NM_004944.1 /DB_XREF = gi: 4826697 /GEN = DNASE1L3
chr3p21.1-3p14.3
Hs.88646
deoxyribonuclease I-like 3
DNASE1L3




/FEA = FLmRNA /CNT = 51 /TID = Hs.88646.0 /TIER = FL /STK = 0 /UG = Hs.88646




/LL = 1776 /DEF = Homo sapiens deoxyribonuclease I-like 3 (DNASE1L3),




mRNA. /PROD = deoxyribonuclease I-like 3 /FL = gb: U56814.1 gb: AF047354.1




gb: NM—


204014_at
NM_001394
gb: NM_001394.2 /DB_XREF = gi: 12707552 /GEN = DUSP4 /FEA = FLmRNA
chr8p12-p11
Hs.417962
dual specificity phosphatase 4
DUSP4




/CNT = 105 /TID = Hs.2359.0 /TIER = FL /STK = 4 /UG = Hs.2359 /LL = 1846




/DEF = Homo sapiens dual specificity phosphatase 4 (DUSP4), mRNA.




/PROD = dual specificity phosphatase 4 /FL = gb: NM_001394.2 gb: BC002671.1 g


204271_s_at
M74921
gb: M74921.1 /DB_XREF = gi: 182275 /GEN = ETs /FEA = FLmRNA /CNT = 130
chr13q22
Hs.82002
endothelin receptor type B
EDNRB




/TID = Hs.82002.0 /TIER = FL /STK = 6 /UG = Hs.82002 /LL = 1910 /DEF = Human




endothelin receptor mRNA, complete cds. /PROD = endothelin receptor




/FL = gb: D90402.1 gb: NM_000115.1 gb: M74921.1


206070_s_at
AF213459
gb: AF213459.1 /DB_XREF = gi: 12003434 /GEN = EPHA3 /FEA = FLmRNA
chr3p11.2
Hs.123642
EPH receptor A3
EPHA3




/CNT = 20 /TID = Hs.123642.0 /TIER = FL /STK = 0 /UG = Hs.123642 /LL = 2042




/DEF = Homo sapiens ephrin receptor EPHA3 complete form (EPHA3) mRNA,




complete cds. /PROD = ephrin receptor EPHA3 complete form /FL = gb: NM


221884_at
BE466525
gb: BE466525 /DB_XREF = gi: 9512223 /DB_XREF = hx9Ab10.x1
chr3q24-q28
Hs.436019
ecotropic viral integration site 1
EVI1




/CLONE = IMAGE: 3195451 /FEA = mRNA /CNT = 52 /TID = Hs.234773.0




/TIER = Stack /STK = 13 /UG = Hs.234773 /UG_TITLE = Homo sapiens cDNA:




FLJ22281 fis, clone HRC03849, highly similar to S69002 human mRNA for




AML1-EVI-1


238877_at
BE674583
gb: BE674583 /DB_XREF = gi: 10035124 /DB_XREF = 7e02h07.x1


Eyes absent homolog 4
EYA4




/CLONE = IMAGE: 3281341 /FEA = EST /CNT = 7 /TID = Hs.102408.0


(Drosophila)




/TIER = ConsEnd /STK = 4 /UG = Hs.102408 /UG_TITLE = ESTs


202771_at
NM_014745
gb: NM_014745.1 /DB_XREF = gi: 7662013 /GEN = KIAA0233 /FEA = FLmRNA
chr16q24.3
Hs.79077
family with sequence similarity 38,
FAM38A




/CNT = 124 /TID = Hs.79077.0 /TIER = FL + Stack /STK = 53 /UG = Hs.79077


member A




/LL = 9780 /DEF = Homo sapiens KIAA0233 gene product (KIAA0233), mRNA.




/PROD = KIAA0233 gene product /FL = gb: D87071.1 gb: NM_014745.1


227475_at
AI676059
gb: AI676059 /DB_XREF = gi: 4876539 /DB_XREF = wc04g08.x1
chr6p25
Hs.297452
forkhead box Q1
FOXQ1




/CLONE = IMAGE: 2314238 /FEA = EST /CNT = 31 /TID = Hs.163900.0




/TIER = Stack /STK = 23 /UG = Hs.163900 /UG_TITLE = ESTs


219764_at
NM_007197
gb: NM_007197.1 /DB_XREF = gi: 6005761 /GEN = FZD10 /FEA = FLmRNA
chr12q24.33
Hs.31664
frizzled homolog 10 (Drosophila)
FZD10




/CNT = 27 /TID = Hs.31664.0 /TIER = FL + Stack /STK = 16 /UG = Hs.31664




/LL = 11211 /DEF = Homo sapiens frizzled (Drosophila) homolog 10 (FZD10),




mRNA. /PROD = frizzled (Drosophila) homolog 10 /FL = gb: AB027464.1 gb: N


227405_s_at
AW340311
gb: AW340311 /DB_XREF = gi: 6836937 /DB_XREF = hc95f03.x1
chr10p11.21
Hs.302634
frizzled homolog 8 (Drosophila)
FZD8




/CLONE = IMAGE: 2907773 /FEA = EST /CNT = 38 /TID = Hs.32659.0




/TIER = Stack /STK = 24 /UG = Hs.32659 /UG_TITLE = ESTs


208463_at
NM_000809
gb: NM_000809.1 /DB_XREF = gi: 4557604 /GEN = GABRA4 /FEA = FLmRNA
chr4p12
Hs.248112
gamma-aminobutyric acid (GABA)
GABRA4




/CNT = 8 /TID = Hs.248112.0 /TIER = FL /STK = 0 /UG = Hs.248112 /LL = 2557


A receptor, alpha 4




/DEF = Homo sapiens gamma-aminobutyric acid (GABA) A receptor, alpha 4




(GABRA4), mRNA. /PROD = gamma-aminobutyric acid A receptor, alpha


218885_s_at
NM_024642
gb: NM_024642.1 /DB_XREF = gi: 13375880 /GEN = FLJ21212
chr9q22.33
Hs.47099
UDP-N-acetyl-alpha-D-
GALNT12




/FEA = FLmRNA /CNT = 70 /TID = Hs.47099.0 /TIER = FL /STK = 0 /UG = Hs.47099


galactosamine: polypeptide N-




/LL = 79695 /DEF = Homo sapiens hypothetical protein FLJ21212 (FLJ21212),


acetylgalactosaminyltransferase 12




mRNA. /PROD = hypothetical protein FLJ21212 /FL = gb: NM_024642.1


(GalNAc-T12)


230360_at
AW006648
gb: AW006648 /DB_XREF = gi: 5855426 /DB_XREF = wt06e01.x1
chr15q21.2
Hs.30484
gliomedin
GLDN




/CLONE = IMAGE: 2506680 /FEA = EST /CNT = 10 /TID = Hs.30484.0




/TIER = Stack /STK = 8 /UG = Hs.30484 /UG_TITLE = ESTs


206355_at
R20102
gb: R20102 /DB_XREF = gi: 774736 /DB_XREF = yg39h06.r1
chr18p11.22-p11.21
Hs.136295
guanine nucleotide binding protein
GNAL




/CLONE = IMAGE: 35031 /FEA = FLmRNA /CNT = 20 /TID = Hs.288642.0


(G protein), alpha activating activity




/TIER = ConsEnd /STK = 0 /UG = Hs.288642 /LL = 2774 /UG_GENE = GNAL


polypeptide, olfactory type




/UG_TITLE = guanine nucleotide binding protein (G protein), alpha activating




activity polypept


227769_at
AI703476
gb: Al703476 /DB_XREF = gi: 4991376 /DB_XREF = we24f08.x1


G protein-coupled receptor 27
GPR27




/CLONE = IMAGE: 2342055 /FEA = EST /CNT = 30 /TID = Hs.250899.1




/TIER = Stack /STK = 17 /UG = Hs.250899 /LL = 3281 /UG_GENE = HSBP1




/UG_TITLE = heat shock factor binding protein 1


206002_at
NM_005756
gb: NM_005756.1 /DB_XREF = gi: 5031732 /GEN = GPR64 /FEA = FLmRNA
chrxp22.13
Hs.421137
G protein-coupled receptor 64
GPR64




/CNT = 24 /TID = Hs.184942.0 /TIER = FL + Stack /STK = 9 /UG = Hs.184942




/LL = 10149 /DEF = Homo sapiens G protein-coupled receptor 64 (GPR64),




mRNA. /PROD = G protein-coupled receptor 64 /FL = gb: NM_005756.1


201348_at
NM_002084
gb: NM_002084.2 /DB_XREF = gi: 6006000 /GEN = GPX3 /FEA = FLmRNA
chr5q23
Hs.386793
glutathione peroxidase 3 (plasma)
GPX3




/CNT = 468 /TID = Hs.172153.0 /TIER = FL + Stack /STK = 256 /UG = Hs.172153




/LL = 2878 /DEF = Homo sapiens glutathione peroxidase 3 (plasma) (GPX3),




mRNA. /PROD = plasma glutathione peroxidase 3 precursor /FL = gb: NM_0


218959_at
NM_017409
gb: NM_017409.1 /DB_XREF = gi: 8393550 /GEN = HOXC10 /FEA = FLmRNA
chr12q13.3
Hs.44276
homeo box C10
HOXC10




/CNT = 63 /TID = Hs.44276.0 /TIER = FL + Stack /STK = 18 /UG = Hs.44276




/LL = 3226 /DEF = Homo sapiens homeo box C10 (HOXC10), mRNA.




/PROD = homeo box C10 /FL = gb: BC001293.1 gb: NM_017409.1




gb: AF255675.1


208937_s_at
D13889
gb: D13889.1 /DB_XREF = gi: 464181 /GEN = Id-1H /FEA = FLmRNA
chr20q11
Hs.410900
inhibitor of DNA binding 1,
ID1




/CNT = 355 /TID = Hs.75424.1 /TIER = FL /STK = 0 /UG = Hs.75424 /LL = 3397


dominant negative helix-loop-helix




/DEF = Human mRNA for Id-1H, complete cds. /PROD = Id-1H


protein




/FL = gb: NM_002165.1 gb: BC000613.1 gb: D13889.1


211430_s_at
M87789
gb: M87789.1 /DB_XREF = gi: 185361 /FEA = FLmRNA /CNT = 1
chr14q32.33
Hs.413826
immunoglobulin heavy locus ///
IGH@ /// IGHG1




/TID = Hs.300697.0 /TIER = FL /STK = 0 /UG = Hs.300697 /LL = 3502


immunoglobulin heavy constant
/// IGHG2 ///




/UG_GENE = IGHG3 /DEF = Human (hybridoma H210) anti-hepatitis A IgG


gamma 1 (G1m marker) ///
IGHG3 /// IGHM




variable region, constant region, complementarity-determining regions


immunoglobulin heavy constant




mRNA, complete


gamma 2 (G2m marker) ///







immunoglobulin heavy constant







gamma 3 (G3m marker) ///







immunoglobulin heavy constant mu


212592_at
AV733266
gb: AV733266 /DB_XREF = gi: 10850811 /DB_XREF = AV733266
chr4q21
Hs.381568
Immunoglobulin J polypeptide,
IGJ




/CLONE = cdAAJG04 /FEA = EST /CNT = 270 /TID = Hs.76325.1 /TIER = Stack


linker protein for immunoglobulin




/STK = 67 /UG = Hs.76325 /LL = 10569 /UG_GENE = SLU7 /UG_TITLE = step II


alpha and mu polypeptides




splicing factor SLU7


209138_x_at
M87790
gb: M87790.1 /DB_XREF = gi: 185363 /FEA = FLmRNA /CNT = 660
chr22q11.2
Hs.458262
Immunoglobulin lambda joining 3
IGLC2




/TID = Hs.181125.0 /TIER = FL + Stack /STK = 584 /UG = Hs.181125 /LL = 3535




/UG_GENE = IGL@ /DEF = Human (hybridoma H210) anti-hepatitis A




immunoglobulin lambda chain variable region, constant region,




complementarity-de


206766_at
AF112345
gb: AF112345.1 /DB_XREF = gi: 6650627 /GEN = ITGA10 /FEA = FLmRNA
chr1q21
Hs.158237
integrin, alpha 10
ITGA10




/CNT = 13 /TID = Hs.158237.0 /TIER = FL /STK = 0 /UG = Hs.158237 /LL = 8515




/DEF = Homo sapiens integrin alpha 10 subunit (ITGA10) mRNA, complete




cds. /PROD = integrin alpha 10 subunit /FL = gb: AF112345.1 gb: NM_0036


214927_at
AL359052
gb: AL359052.1 /DB_XREF = gi: 8518175 /FEA = mRNA /CNT = 13


Integrin, beta-like 1 (with EGF-like
ITGBL1




/TID = Hs.311054.0 /TIER = ConsEnd /STK = 5 /UG = Hs.311054 /DEF = Homo


repeat domains)




sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422.




/PROD = ITGBL1, integrin beta-like 1


221841_s_at
BF514079
gb: BF514079 /DB_XREF = gi: 11599258 /DB_XREF = UI-H-BW1-amw-b-08-
chr9q31
Hs.376206
Kruppel-like factor 4 (gut)
KLF4




0-UI.s1 /CLONE = IMAGE: 3071198 /FEA = EST /CNT = 61 /TID = Hs.7934.1




/TIER = Stack /STK = 11 /UG = Hs.7934 /LL = 9314 /UG_GENE = KLF4




/UG_TITLE = Kruppel-like factor 4 (gut)


204249_s_at
NM_005574
gb: NM_005574.2 /DB_XREF = gi: 6633806 /GEN = LMO2 /FEA = FLmRNA
chr11p13
Hs.283063
LIM domain only 2 (rhombotin-like
LMO2




/CNT = 93 /TID = Hs.184585.0 /TIER = FL + Stack /STK = 42 /UG = Hs.184585


1)




/LL = 4005 /DEF = Homo sapiens LIM domain only 2 (rhombotin-like 1) (LMO2),




mRNA. /PROD = LIM domain only 2 /FL = gb: NM_005574.2


228653_at
AI700341
gb: AI700341 /DB_XREF = gi: 4988241 /DB_XREF = wd06e10.x1


SAM domain containing 1
LOC389432




/CLONE = IMAGE: 2327370 /FEA = EST /CNT = 23 /TID = Hs.110406.0




/TIER = Stack /STK = 14 /UG = Hs.110406 /UG_TITLE = ESTs


206953_s_at
NM_012302
gb: NM_012302.1 /DB_XREF = gi: 6912463 /GEN = KIAA0786 /FEA = FLmRNA
chr1p31.1
Hs.24212
latrophilin 2
LPHN2




/CNT = 8 /TID = Hs.24212.0 /TIER = FL /STK = 0 /UG = Hs.24212 /LL = 23266




/DEF = Homo sapiens latrophilin (KIAA0786), mRNA. /PROD = latrophilin




/FL = gb: NM_012302.1 gb: AF104939.1


231781_s_at
AK021919
gb: AK021919.1 /DB_XREF = gi: 10433216 /FEA = FLmRNA /CNT = 13
chr3p21.31
Hs.380055
leucine rich repeat containing 2
LRRC2




/TID = Hs.125790.2 /TIER = ConsEnd /STK = 0 /UG = Hs.125790 /LL = 79442




/UG_GENE = LRRC2 /UG_TITLE = leucine-rich repeat-containing 2




/DEF = Homo sapiens cDNA FLJ11857 fis, clone HEMBA1006807, moderately




similar to H


210302_s_at
AF262032
gb: AF262032.1 /DB_XREF = gi: 9964006 /GEN = MAB21L2 /FEA = FLmRNA
chr4q31
Hs.251390
mab-21-like 2 (C. elegans)
MAB21L2




/CNT = 17 /TID = Hs.251390.0 /TIER = FL /STK = 0 /UG = Hs.251390 /LL = 10586




/DEF = Homo sapiens MAB21L2 protein (MAB21L2) mRNA, complete cds.




/PROD = MAB21L2 protein /FL = gb: AF262032.1 gb: NM_006439.2


202291_s_at
NM_000900
gb: NM_000900.1 /DB_XREF = gi: 4505178 /GEN = MGP /FEA = FLmRNA
chr12p13.1-p12.3
Hs.365706
matrix Gla protein
MGP




/CNT = 227 /TID = Hs.279009.0 /TIER = FL + Stack /STK = 84 /UG = Hs.279009




/LL = 4256 /DEF = Homo sapiens matrix Gla protein (MGP), mRNA.




/PROD = matrix Gla protein /FL = gb: NM_000900.1 gb: M58549.1


205680_at
NM_002425
gb: NM_002425.1 /DB_XREF = gi: 4505204 /GEN = MMP10 /FEA = FLmRNA
chr11q22.3
Hs.2258
matrix metallopeptidase 10
MMP10




/CNT = 29 /TID = Hs.2258.0 /TIER = FL /STK = 6 /UG = Hs.2258 /LL = 4319


(stromelysin 2)




/DEF = Homo sapiens matrix metalloproteinase 10 (stromelysin 2) (MMP10),




mRNA. /PROD = matrix metalloproteinase 10 preproprotein /FL = gb: BC002


205330_at
NM_002430
gb: NM_002430.1 /DB_XREF = gi: 4505222 /GEN = MN1 /FEA = FLmRNA
chr22q12.1
Hs.268515
meningioma (disrupted in balanced
MN1




/CNT = 43 /TID = Hs.268515.0 /TIER = FL + Stack /STK = 15 /UG = Hs.268515


translocation) 1




/LL = 4330 /DEF = Homo sapiens meningioma (disrupted in balanced




translocation) 1 (MN1), mRNA. /PROD = meningioma 1 /FL = gb: NM_002430.1


213924_at
BF476502
gb: BF476502 /DB_XREF = gi: 11547329 /DB_XREF = naa27a03.x1


Metallophosphoesterase 1
MPPE1




/CLONE = IMAGE: 3255844 /FEA = EST /CNT = 16 /TID = Hs.154145.3




/TIER = Stack /STK = 9 /UG = Hs.154145 /LL = 65258 /UG_GENE = FLJ11585




/UG_TITLE = hypothetical protein FLJ11585


212096_s_at
AL096842
gb: AL096842.1 /DB_XREF = gi: 5524930 /FEA = mRNA /CNT = 243
chr8p22
Hs.7946
mitochondrial tumor suppressor 1
MTUS1




/TID = Hs.7946.0 /TIER = Stack /STK = 99 /UG = Hs.7946 /LL = 57509




/UG_GENE = KIAA1288 /UG_TITLE = KIAA1288 protein /DEF = Homo sapiens




mRNA; cDNA DKFZp586D1519 (from clone DKFZp586D1519).


217800_s_at
NM_030571
gb: NM_030571.1 /DB_XREF = gi: 13386479 /GEN = MGC10924
chr5q31.3
Hs.9788
Nedd4 family interacting protein 1
NDFIP1




/FEA = FLmRNA /CNT = 361 /TID = Hs.9788.0 /TIER = FL + Stack /STK = 152




/UG = Hs.9788 /LL = 80762 /DEF = Homo sapiens hypothetical protein




MGC10924 similar to Nedd4 WW-binding protein 5 (MGC10924), mRNA.




/PROD = hypothetical


223315_at
AF278532
gb: AF278532.1 /DB_XREF = gi: 11120047 /FEA = FLmRNA /CNT = 97
chr12q22-q23
Hs.102541
netrin 4
NTN4




/TID = Hs.102541.0 /TIER = FL + Stack /STK = 49 /UG = Hs.102541 /LL = 59277




/UG_GENE = NTN4 /DEF = Homo sapiens beta-netrin mRNA, complete cds.




/PROD = beta-netrin /FL = gb: AF119916.1 gb: AF297711.1 gb: NM_021229.1




gb: AF27


236088_at
AV723308
gb: AV723308 /DB_XREF = gi: 10826596 /DB_XREF = AV723308
chr1p13.3
Hs.111224
netrin G1
NTNG1




/CLONE = HTBBFC07 /FEA = EST /CNT = 11 /TID = Hs.171136.0




/TIER = ConsEnd /STK = 2 /UG = Hs.171136 /UG_TITLE = ESTs


212582_at
AL049923
gb: AL049923.1 /DB_XREF = gi: 4884169 /FEA = mRNA /CNT = 140
chr12q14
Hs.109694
oxysterol binding protein-like 8
OSBPL8




/TID = Hs.109694.0 /TIER = Stack /STK = 25 /UG = Hs.109694 /LL = 57601




/UG_GENE = KIAA1451 /UG_TITLE = KIAA1451 protein /DEF = Homo sapiens




mRNA; cDNA DKFZp564E2282 (from clone DKFZp564E2282).


223435_s_at
AI268404
gb: AI268404 /DB_XREF = gi: 3887571 /DB_XREF = qm05e10.x1
chr5q31
Hs.247734
protocadherin alpha 9 ///
PCDHA9 ///




/CLONE = IMAGE: 1880970 /FEA = FLmRNA /CNT = 73 /TID = Hs.167399.0


protocadherin alpha subfamily C, 2
PCDHAC2 ///




/TIER = Stack /STK = 13 /UG = Hs.167399 /LL = 56143 /UG_GENE = PCDHA5


/// protocadherin alpha subfamily C,
PCDHAC1 ///




/UG_TITLE = protocadherin alpha 5 /FL = gb: NM_018908.1 gb: AF152313.1


1 /// protocadherin alpha 13 ///
PCDHA13 ///







protocadherin alpha 12 ///
PCDHA12 ///







protocadherin alpha 11 ///
PCDHA11 ///







protocadherin alpha 10 ///
PCDHA10 ///







protocadherin alpha 8 ///
PCDHA8 ///







protocadherin alpha 7 ///
PCDHA7 ///







protocadherin alpha 6 ///
PCDHA6 ///







protocadherin alpha 5 ///
PCDHA5 ///







protocadherin alpha 4 ///
PCDHA4 ///







protocadherin alpha 3 ///
PCDHA3 ///







protocadherin alpha 2 ///
PCDHA2 ///







protocadherin alpha 1
PCDHA1


1558680_s_at
BQ894022
gb: BQ894022 /DB_XREF = gi: 22286036
chr2q32.1
Hs.416061
phosphodiesterase 1A,
PDE1A




/DB_XREF = AGENCOURT_8122690 /CLONE = IMAGE: 6180918


calmodulin-dependent




/TID = Hs2.383511.1 /CNT = 7 /FEA = mRNA /TIER = ConsEnd /STK = 0




/UG = Hs.383511 /UG_TITLE = Homo sapiens HSPDE1A mRNA for




calmodulin-dependent phosphodiesterase, partial cds, N-terminal


208510_s_at
NM_015869
gb: NM_015869.1 /DB_XREF = gi: 7705548 /GEN = PPARG /FEA = FLmRNA
chr3p25
Hs.387667
peroxisome proliferative
PPARG




/CNT = 4 /TID = Hs.100724.1 /TIER = FL /STK = 0 /UG = Hs.100724 /LL = 5468


activated receptor, gamma




/DEF = Homo sapiens peroxisome proliferative activated receptor, gamma




(PPARG), mRNA. /PROD = peroxisome proliferative activated receptorg


206007_at
NM_005807
gb: NM_005807.1 /DB_XREF = gi: 5031924 /GEN = PRG4 /FEA = FLmRNA
chr1q25-q31
Hs.432458
proteoglycan 4
PRG4




/CNT = 26 /TID = Hs.218791.0 /TIER = FL /STK = 0 /UG = Hs.218791 /LL = 10216




/DEF = Homo sapiens proteoglycan 4, (megakaryocyte stimulating factor,




articular superficial zone protein) (PRG4), mRNA. /PROD = megakary


206805_at
NM_006080
gb: NM_006080.1 /DB_XREF = gi: 5174672 /GEN = SEMA3A /FEA = FLmRNA
chr7p12.1
Hs.252451
sema domain, immunoglobulin
SEMA3A




/CNT = 12 /TID = Hs.2414.0 /TIER = FL /STK = 0 /UG = Hs.2414 /LL = 10371


domain (Ig), short basic domain,




/DEF = Homo sapiens sema domain, immunoglobulin domain (Ig), short basic


secreted, (semaphorin) 3A




domain, secreted, (semaphorin) 3A (SEMA3A), mRNA. /PROD = sema dom


206941_x_at
NM_012431
gb: NM_012431.1 /DB_XREF = gi: 6912649 /GEN = SEMA3E /FEA = FLmRNA
chr7q21.11
Hs.528721
sema domain, immunoglobulin
SEMA3E




/CNT = 7 /TID = Hs.212414.0 /TIER = FL /STK = 0 /UG = Hs.212414 /LL = 9723


domain (Ig), short basic domain,




/DEF = Homo sapiens sema domain, immunoglobulin domain (Ig), short basic


secreted, (semaphorin) 3E




domain, secreted, (semaphorin) 3E (SEMA3E), mRNA. /PROD = sema d


226492_at
AL036088
gb: AL036088 /DB_XREF = gi: 5405713 /DB_XREF = DKFZp564J0223_s1
chr15q21.1
Hs.191098
sema domain, transmembrane
SEMA6D




/CLONE = DKFZp564J0223 /FEA = mRNA /CNT = 49 /TID = Hs.191098.0


domain (TM), and cytoplasmic




/TIER = Stack /STK = 19 /UG = Hs.191098 /LL = 57618 /UG_GENE = KIAA1479


domain, (semaphorin) 6D




/UG_TITLE = KIAA1479 protein


223121_s_at
AW003584
gb: AW003584 /DB_XREF = gi: 5850500 /DB_XREF = wq98h04.x1
chr4q31.3
Hs.31386
secreted frizzled-related protein 2
SFRP2




/CLONE = IMAGE: 2480119 /FEA = FLmRNA /CNT = 207 /TID = Hs.31386.0




/TIER = Stack /STK = 26 /UG = Hs.31386 /LL = 6423 /UG_GENE = SFRP2




/UG_TITLE = secreted frizzled-related protein 2 /FL = gb: AF311912.1


206634_at
NM_005413
gb: NM_005413.1 /DB_XREF = gi: 4885596 /GEN = SIX3 /FEA = FLmRNA
chr2p16-p21
Hs.227277
sine oculis homeobox homolog 3
SIX3




/CNT = 14 /TID = Hs.227277.0 /TIER = FL /STK = 1 /UG = Hs.227277 /LL = 6496


(Drosophila)




/DEF = Homo sapiens sine oculis homeobox (Drosophila) homolog 3 (SIX3),




mRNA. /PROD = sine oculis homeobox (Drosophila) homolog 3 /FL = gb: N


223044_at
AL136944
gb: AL136944.1 /DB_XREF = gi: 12053382 /GEN = DKFZp586J0624
chr2q32
Hs.409875
solute carrier family 40 (iron-
SLC40A1




/FEA = FLmRNA /CNT = 341 /TID = Hs.5944.0 /TIER = FL + Stack /STK = 188


regulated transporter), member 1




/UG = Hs.5944 /LL = 30061 /DEF = Homo sapiens mRNA; cDNA




DKFZp586J0624 (from clone DKFZp586J0624); complete cds.




/PROD = hypothetical protein /FL = gb:


209921_at
AB040875
gb: AB040875.1 /DB_XREF = gi: 13516845 /GEN = hxCT /FEA = FLmRNA
chr4q28-q32
Hs.6682
solute carrier family 7, (cationic
SLC7A11




/CNT = 45 /TID = Hs.6682.1 /TIER = FL + Stack /STK = 33 /UG = Hs.6682


amino acid transporter, y+ system)




/LL = 23657 /DEF = Homo sapiens hxCT mRNA for cystineglutamate


member 11




exchanger, complete cds. /PROD = cystineglutamate exchanger




/FL = gb: AB040875.1


222784_at
AJ249900
gb: AJ249900.1 /DB_XREF = gi: 10432430 /GEN = smoc1 /FEA = FLmRNA
chr14q24.2
Hs.14144
SPARC related modular calcium
SMOC1




/CNT = 89 /TID = Hs.14144.0 /TIER = Stack /STK = 33 /UG = Hs.14144 /LL = 64093


binding 1




/DEF = Homo sapiens mRNA for secreted modular calcium-binding protein




(smoc1 gene). /PROD = secreted modular calcium-binding protein /


227752_at
AA005105
gb: AA005105 /DB_XREF = gi: 1448894 /DB_XREF = zh96f09.s1

Hs.425023
serine palmitoyltransferase, long
SPTLC2L




/CLONE = IMAGE: 429161 /FEA = EST /CNT = 38 /TID = Hs.18441.0 /TIER = Stack


chain base subunit 2-like




/STK = 11 /UG = Hs.18441 /UG_TITLE = ESTs


(aminotransferase 2)


203000_at
BF967657
gb: BF967657 /DB_XREF = gi: 12334872 /DB_XREF = 602287358T1
chr8q21.13
Hs.90005
stathmin-like 2
STMN2




/CLONE = IMAGE: 4374495 /FEA = FLmRNA /CNT = 403 /TID = Hs.90005.0




/TIER = Stack /STK = 18 /UG = Hs.90005 /LL = 11075 /UG_GENE = SCGN10




/UG_TITLE = superiorcervical ganglia, neural specific 10




/FL = gb: NM_007029.1 gb: D50375.


211276_at
AF063606
gb: AF063606.1 /DB_XREF = gi: 12002041 /FEA = FLmRNA /CNT = 1


transcription elongation factor A
TCEAL2




/TID = Hs.17481.0 /TIER = FL /STK = 0 /UG = Hs.17481 /DEF = Homo sapiens


(SII)-like 2




brain my048 protein mRNA, complete cds. /PROD = brain my048 protein




/FL = gb: AF063606.1


206243_at
NM_003256
gb: NM_003256.1 /DB_XREF = gi: 4507514 /GEN = TIMP4 /FEA = FLmRNA
chr3p25
Hs.190787
TIMP metallopeptidase inhibitor 4
TIMP4




/CNT = 25 /TID = Hs.190787.0 /TIER = FL + Stack /STK = 16 /UG = Hs.190787




/LL = 7079 /DEF = Homo sapiens tissue inhibitor of metalloproteinase 4




(TIMP4), mRNA. /PROD = tissue inhibitor of metalloproteinase 4precurso


203661_s_at
BC002660
gb: BC002660.1 /DB_XREF = gi: 12803650 /FEA = FLmRNA /CNT = 110
chr9q22.3
Hs.374849
tropomodulin 1
TMOD1




/TID = Hs.170453.0 /TIER = FL + Stack /STK = 19 /UG = Hs.170453 /LL = 7111




/UG_GENE = TMOD /DEF = Homo sapiens, tropomodulin, clone MGC: 3643,




mRNA, complete cds. /PROD = tropomodulin /FL = gb: NM_003275.1




gb: M77016.1 gb:


221085_at
NM_005118
gb: NM_005118.1 /DB_XREF = gi: 4827031 /GEN = TNFSF15 /FEA = FLmRNA
chr9q32
Hs.241382
tumor necrosis factor (ligand)
TNFSF15




/CNT = 2 /TID = Hs.241382.0 /TIER = FL /STK = 0 /UG = Hs.241382 /LL = 9966


superfamily, member 15




/DEF = Homo sapiens tumor necrosis factor (ligand) superfamily, member 15




(TNFSF15), mRNA. /PROD = tumor necrosis factor (ligand) superf


202341_s_at
AA149745
gb: AA149745 /DB_XREF = gi: 1720818 /DB_XREF = zo02h04.s1
chr4q31.3
Hs.435734
tripartite motif-containing 2
TRIM2




/CLONE = IMAGE: 566551 /FEA = FLmRNA /CNT = 143 /TID = Hs.12372.0




/TIER = Stack /STK = 18 /UG = Hs.12372 /LL = 23321 /UG_GENE = KIAA0517




/UG_TITLE = tripartite motif protein TRIM2 /FL = gb: AF220018.1




gb: NM_015271.1


242162_at
AA904430
gb: AA904430 /DB_XREF = gi: 3039553 /DB_XREF = ok07f12.s1
chr2q36.3
Hs.424594
WD repeat domain 69
WDR69




/CLONE = IMAGE: 1507151 /FEA = EST /CNT = 5 /TID = Hs.122049.0




/TIER = ConsEnd /STK = 1 /UG = Hs.122049 /UG_TITLE = ESTs, Weakly similar




to T2D4_HUMAN TRANSCRIPTION INITIATION FACTOR TFIID 100 KDA




SUBUNIT (H. sapiens)


206373_at
NM_003412
gb: NM_003412.1 /DB_XREF = gi: 4507970 /GEN = ZIC1 /FEA = FLmRNA


Zic family member 1 (odd-paired
ZIC1




/CNT = 19 /TID = Hs.41154.0 /TIER = FL /STK = 7 /UG = Hs.41154 /LL = 7545


homolog, Drosophila)




/DEF = Homo sapiens Zic family member 1 (odd-paired Drosophila homolog)




(ZIC1), mRNA. /PROD = Zic family member 1 (odd-paired Drosophilahomo


210910_s_at
BC000487
gb: BC000487.1 /DB_XREF = gi: 12653432 /FEA = FLmRNA /CNT = 3
chr7q11.23
Hs.296380
zona pellucida glycoprotein 3
ZP3 /// POMZP3




/TID = Hs.296380.1 /TIER = FL /STK = 0 /UG = Hs.296380 /LL = 22932


(sperm receptor) /// POM (POM121




/UG_GENE = POMZP3 /DEF = Homo sapiens, Similar to POM (POM121 rat


homolog, rat) and ZP3 fusion




homolog) and ZP3 fusion protein, clone MGC: 8359, mRNA, complete cds.




/PROD = Similar







collomin
COLM









FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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## Claims

1. A method of diagnosing or monitoring rheumatoid arthritis in a subject, comprising the steps:
determining the activation state of a circulating CD3-CD45+collagen+ fibrocyte from a sample from the subject; and
comparing the activation state of the fibrocyte from the sample with a control;
wherein the activation state of the fibrocyte is determined by measuring the phosphorylation levels of signaling molecules, and
wherein an increase in the activation state of the fibrocyte as compared to the control is indicative of rheumatoid arthritis.

2. The method according to claim 1, wherein the signaling molecules comprise STAT5.

3. The method according to claim 1, wherein the circulating fibrocyte further stains positive for CD14, CD34 and prolyl 4-hydroxylase.

4. A method of identifying a substance to treat rheumatoid arthritis, comprising the steps:
(a) determining the activation state of circulating CD3-CD45+collagen+ fibrocytes from a sample from a subject administered the substance; and
(b) comparing the activation state of the fibrocyte from the sample with a control;
wherein the activation state of the fibrocyte is determined by measuring the phosphorylation levels of signaling molecules, and
wherein a decrease in the activation state of the fibrocyte as compared to the control is indicative of a substance to treat rheumatoid arthritis.

5. The method according to claim 4, wherein the signaling molecules comprise STAT5.

6. The method according to claim 4, wherein the circulating fibrocytes further stains positive for CD14, CD34 and prolyl 4-hydroxylase.