Systemic Lupus Erythematosus

Abstract

This document relates to methods and materials involved in diagnosing SLE. For example, this document provides arrays for detecting polypeptides that can be used to diagnose SLE in a mammal. In addition, methods and materials for assessing SLE activity, determining the likelihood of experiencing active SLE, and detecting SLE treatment effectiveness are provided herein.

Description

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in diagnosing systemic lupus erythematosus (SLE). For example, this document relates to methods and materials involved in diagnosing SLE, assessing a mammal's susceptibility to develop SLE, and assessing SLE activity.

2. Background Information

SLE is a chronic, inflammatory autoimmune disease characterized by the production of autoantibodies having specificity for a wide range of self-antigens. SLE autoantibodies mediate organ damage by directly binding to host tissues and by forming immune complexes that deposit in vascular tissues and activate immune cells. Organs targeted in SLE include the skin, kidneys, vasculature, joints, various blood elements, and the central nervous system (CNS). The severity of disease, the spectrum of clinical involvement, and the response to therapy vary widely among patients. This clinical heterogeneity makes it challenging to diagnose and manage lupus.

SUMMARY

This document relates to methods and materials involved in diagnosing SLE. For example, this document relates to methods and materials involved in diagnosing SLE, assessing a mammal's susceptibility to develop SLE, and assessing SLE activity. For example, this document provides arrays for detecting polypeptides that can be used to diagnose SLE in a mammal. Such arrays can allow clinicians to diagnose SLE based on a determination of the levels of many polypeptides that are differentially regulated in SLE patients as compared to healthy controls. This document also provides methods and materials that can be used to assess SLE activity. Assessing SLE activity can allow clinicians to identify patients with active SLE. In addition, this document provides methods and materials that can be used to assess the likelihood that a patient will experience active SLE. For example, a patient found to have serum containing one or more polypeptides listed in Table 3 at a level that is greater than or less than the average level observed in control serum can be classified as being likely to experience active SLE. This document also provides methods and materials that can be used to determine whether or not a mammal responds to an SLE treatment. For example, patients receiving an SLE treatment (e.g., an anti-IFN treatment) who are found have serum that no longer contains one or more IFN-regulated chemokines at a level greater than or less than the average level observed in control serum can be classified as responding to that SLE treatment.

Typically, a diagnosis of SLE can be made on the basis of 11 criteria defined by the American College of Rheumatology (ACR). These criteria include malar rash, discoid rash, photosensitivity, oral ulcers, arthritis, serositis, renal disorder, neurologic disorder, hematologic disorder, immunologic disorder, and antinuclear antibody (Tan et al. (1982) Arthritis Rheum 25:1271-1277). A mammal (e.g., a human) can be clinically diagnosed with SLE if he or she meets at least four of the eleven criteria.

This document is based, in part, on the discovery of polypeptides that are differentially regulated between SLE patients and healthy controls. This document also is based, in part, on the discovery that the serum levels of polypeptides can be used to distinguish mammals with SLE from healthy mammals. For example, the serum levels for the polypeptides listed in Table 2 can be assessed to diagnose SLE.

For the purpose of this document, the term “activity signature 1” as used herein refers to a serum polypeptide profile where one or more (e.g., two, three, four, five, six, seven, eight nine, ten, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or more) of the polypeptides listed in Table 2 are present at a level greater than or less than the level observed in control serum from a control mammal. In some cases, the activity signature 1 can be a serum polypeptide profile where 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent of the polypeptides listed in Table 2 are present at a level greater than or less than the level observed in control serum from a control mammal. The term “activity signature 2” as used herein refers to a serum polypeptide profile where one or more (e.g., two, three, four, five, six, seven, eight nine, ten, 15, 20, 25, 27, or more) of the polypeptides listed in Table 3 are present at a level greater than or less than the level observed in control serum from a control mammal. In some cases, the activity signature 2 can be a serum polypeptide profile where 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent of the polypeptides listed in Table 3 are present at a level greater than or less than the level observed in control serum from a control mammal. The term “activity signature 3” as used herein refers to a serum polypeptide profile where one or more (e.g., two, three, four, five, six, seven, eight nine, ten, 15, 18, or more) of the polypeptides listed in Table 4 are present at a level greater than or less than the level observed in control serum from a control mammal. In some cases, the activity signature 3 can be a serum polypeptide profile where 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent of the polypeptides listed in Table 4 are present at a level greater than or less than the level observed in control serum from a control mammal. The term “activity signature 4” as used herein refers to a serum polypeptide profile where one or more (e.g., two, three, four, five, six, or more) polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides are present at a level greater than or less than the level observed in control serum from a control mammal. In some cases, the activity signature 4 can be a serum polypeptide profile where 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent of the polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11polypeptides are present at a level greater than or less than the level observed in control serum from a control mammal.

In general, one aspect of this document features a method for identifying a mammal having systemic lupus erythematosus. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal comprises a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4, and (b) classifying the mammal as having systemic lupus erythematosus if the serum comprises the signature and classifying the mammal as not having systemic lupus erythematosus if the serum does not comprise the signature. The mammal can be a human. The method can include determining whether or not the serum comprises the activity signature 1. The method can include determining whether or not the serum comprises the activity signature 2. The method can include determining whether or not the serum comprises the activity signature 3. The method can include determining whether or not the serum comprises the activity signature 4.

In another embodiment, this document features a method for identifying a mammal having systemic lupus erythematosus. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal contains one or more of the polypeptides listed in Table 2, 3, or 4 at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from one or more control mammals, and (b) classifying the mammal as having systemic lupus erythematosus if the serum contains the one or more polypeptides and classifying the mammal as not having systemic lupus erythematosus if the serum does not contain the one or more polypeptides. The mammal can be a human. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 2. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 3. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 4.

In another embodiment, this document features a method for identifying a mammal having systemic lupus erythematosus. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal contains one or more of the polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from one or more control mammals, and (b) classifying the mammal as having systemic lupus erythematosus if the serum contains the one or more polypeptides and classifying the mammal as not having systemic lupus erythematosus if the serum does not contain the one or more polypeptides. The mammal can be a human.

In another embodiment, this document features a method for assessing systemic lupus erythematosus disease activity. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal comprises a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4, and (b) classifying the mammal as having active systemic lupus erythematosus disease if the serum comprises the signature and classifying the mammal as not having active systemic lupus erythematosus disease if the serum does not comprise the signature. The mammal can be a human. The method can include determining whether or not the serum comprises the activity signature 1. The method can include determining whether or not the serum comprises the activity signature 2. The method can include determining whether or not the serum comprises the activity signature 3. The method can include determining whether or not the serum comprises the activity signature 4.

In another embodiment, this document features a method for assessing systemic lupus erythematosus disease activity. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal contains one or more of the polypeptides listed in Table 2, 3, or 4 at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from one or more control mammals, and (b) classifying the mammal as having active systemic lupus erythematosus disease if the serum contains the one or more polypeptides and classifying the mammal as not having active systemic lupus erythematosus disease if the serum does not contain the one or more polypeptides. The mammal can be a human. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 2. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 3. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 4.

In another embodiment, this document features a method for assessing systemic lupus erythematosus disease activity. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal contains one or more of the potypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides, and (b) classifying the mammal as having active systemic lupus erythematosus disease if the serum contains the one or more polypeptides and classifying the mammal as not having active systemic lupus erythematosus disease if the serum does not contain the one or more polypeptides. The mammal can be a human.

In another embodiment, this document features a method for determining whether or not a mammal is likely to experience active systemic lupus erythematosus disease. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal comprises a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4, and (b) classifying the mammal as being likely to experience the active systemic lupus erythematosus disease if the serum comprises the signature and classifying the mammal as not being likely to experience the active systemic lupus erythematosus disease if the serum does not comprise the signature. The mammal can be a human. The method can include determining whether or not the serum comprises the activity signature 1. The method can include determining whether or not the serum comprises the activity signature 2. The method can include determining whether or not the serum comprises the activity signature 3. The method can include determining whether or not the serum comprises the activity signature 4.

In another embodiment, this document features a method for determining whether or not a mammal is likely to experience active systemic lupus erythematosus disease. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal contains one or more of the polypeptides listed in Table 2, 3, or 4 at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from one or more control mammals, and (b) classifying the mammal as being likely to experience the active systemic lupus erythematosus disease if the serum contains the one or more polypeptides and classifying the mammal as not being likely to experience the active systemic lupus erythematosus disease if the serum does not contain the one or more polypeptides. The mammal can be a human. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides)listed in Table 2. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 3. The method can include determining whether or not the serum contains one or more polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 4.

In another embodiment, this document features a method for determining whether or not a mammal is likely to experience active systemic lupus erythematosus disease. The method comprises, or consists essentially of, (a) determining whether or not serum from a mammal contains one or more of the polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from one or more control mammals, and (b) classifying the mammal as being likely to experience the active systemic lupus erythematosus disease if the serum contains the one or more polypeptides and classifying the mammal as not being likely to experience the active systemic lupus erythematosus disease if the serum does not contain the one or more polypeptides. The mammal can be a human.

In another embodiment, this document features a method for assessing effectiveness of a treatment for systemic lupus erythematosus disease. The method comprises, or consists essentially of, determining whether or not a mammal having systemic lupus erythematosus disease and having received a treatment for the systemic erythematosus disease comprises serum having a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4 at a level less than the level present in an earlier obtained serum sample from the mammal, where the presence of the serum indicates that the treatment is effective. The mammal can be a human. The method can include determining whether or not the serum comprises the activity signature 1. The method can include determining whether or not the serum comprises the activity signature 2. The method can include determining whether or not the serum comprises the activity signature 3. The method can include determining whether or not the serum comprises the activity signature 4.

In another embodiment, this document features a method for assessing effectiveness of a treatment for systemic lupus erythematosus disease. The method comprises, or consists essentially of, determining whether or not a mammal having systemic lupus crythematosus disease and having received a treatment for the systemic erythematosus disease has serum containing one or more of the polypeptides listed in Table 2, 3, or 4 at a level less than the level present in an earlier obtained serum sample from the mammal, where the presence of the serum indicates that the treatment is effective. The mammal can be a human. The method can include determining whether or not the serum contains one or more of the polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 2. The method can include determining whether or not the serum contains one or more of the polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 3. The method can include determining whether or not the serum contains one or more of the polypeptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides) listed in Table 4.

In yet another aspect, this document features a method for assessing effectiveness of a treatment for systemic lupus erythematosus disease. The method comprises, or consists essentially of, determining whether or not a mammal having systemic lupus erythematosus disease and having received a treatment for the systemic erythematosus disease has serum containing one or more of the polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level less than the level present in an earlier obtained serum sample from the mammal, where the presence of the serum indicates that the treatment is effective. The mammal can be a human.

In yet another aspect, this document features an array for detecting polypeptides. The array comprises, or consists essentially of, at least 5 polypeptides (e.g., at least 6, 7, 8, 9, 10, 15, 20, 30, or more polypeptides) capable of detecting polypeptides, wherein each of the at least 5 polypeptides has a different amino acid sequence. The array can contain at least 50 polypeptides capable of detecting polypeptides, wherein each of the at least 50 polypeptides has a different amino acid sequence. The polypeptides capable of detecting polypeptides can be antibodies or antibody fragments. An antibody or antibody fragment can be capable of detecting (e.g., via binding at typical antibody affinity) a polypeptide listed in Table 2, 3, or 4. The array can contain glass.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting IFN gene expression scores for IFN-hi SLE, IFN-lo SLE and control subjects. Whole blood gene expression microarrays were used to identify 82 type I IFN-regulated genes that distinguished 81 SLE cases from 42 controls. These genes were used to derive a normalized IFN gene score. Plotted are IFN gene scores for 15 IFN-hi SLE (mean±SD, 41.0±4.8), 15 IFN-lo SLE (14.4±2.8), and 15 controls (12.1±1.9).

FIG. 2 is a set of graphs plotting serum levels of CCL2 (upper panel) or CXCL9 (lower panel) measured using Luminex bead-based immunoassays against the levels measured using antibody arrays. Linear regression analysis was performed. The units are pg/mL.

FIG. 3 is a graph plotting correlation coefficients between serum polypeptide levels and blood gene expression levels. Linear regression analysis was used to determine the correlations between serum polypeptide levels and gene expression levels in whole blood measured concurrently using Affymetrix microarrays. P<0.05 thresholds are shown by dotted lines.

FIG. 4 is a graph plotting chemokine polypeptide scores for 15 IFN-hi SLE (mean±SD=2.8±1.2), 15 IFN-lo SLE(1.4±0.5) and 15 controls (1.0±0.2).

DETAILED DESCRIPTION

This document provides methods and materials involved in diagnosing SLE, such as methods and materials involved in diagnosing SLE and assessing a mammal's susceptibility to develop SLE. For example, this document provides arrays for detecting polypeptides that can be used to diagnose SLE in a mammal. Such arrays can allow clinicians to diagnose SLE based on a determination of, for example, serum levels of many polypeptides that are differentially expressed. In addition, the methods and materials provided herein can be used to assess SLE activity, determine the likelihood of experiencing active SLE, and detect SLE treatment effectiveness.

As described herein, this document provides methods for diagnosing a mammal (e.g., a human) as having SLE. In some embodiments, a mammal can be diagnosed as having SLE if it is determined that a sample from the mammal (e.g., a urine or serum sample) contains one or more of the polypeptides listed in Table 2, 3, or 4 at a level that is greater than or less than the average level of the same one or more polypeptides observed in control sample obtained from control mammals. In some cases, a mammal can be diagnosed as having SLE if it is determined that a sample from the mammal (e.g., the mammal's serum) contains one or more of the polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is greater than the average level of the same one or more polypeptides observed in control sample (e.g., control serum) obtained from control mammals.

The mammal can be any mammal such as a human, dog, mouse, or rat. Any method can be used to obtain serum for evaluation. For example, a sample such as serum can be obtained by peripheral venipuncture and evaluated to determine if it contains (1) one or more of the polypeptides listed in Table 2 at a level that is greater than or less than the average level observed in control serum, (2) one or more of the polypeptides listed in Table 3 at a level that is greater than or less than the average level observed in control serum, (3) one or more of the polypeptides listed in Table 4 at a level that is greater than or less than the average level observed in control serum, or (4) one or more of the polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is greater than the average level observed in control serum. The level of any number of polypeptides listed in Table 2, 3, or 4 can be evaluated to diagnose SLE. For example, the level of one or more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, or more than 30) of the polypeptides listed in Table 2, 3, or 4 can be used. Examples of polypeptide combinations that can be used include, without limitation, CCL19 and CXCL9 polypeptides; ACE, IP10, IL6, and MMP7 polypeptides; IL6, IL15, IL18, IL2SRA, and MMP7 polypeptides; and CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides. It will be appreciated that urine samples can be used in place of serum samples for the methods and materials described herein and that urine samples can be obtained using standard urine collection techniques. In some cases, both serum and urine samples can be used as described herein.

The serum level can be greater than or less than the average level observed in control serum obtained from control mammals. Typically, a polypeptide can be classified as being present at a level that is greater than or less than the average level observed in control serum if the levels differ by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or more percent. In some cases, a polypeptide can be classified as being present at a level that is greater than or less than the average level observed in control serum if the levels differ by greater than 1-fold (e.g., 1.5-fold, 2-fold, 3-fold, or more than 3-fold). Control serum typically is of the same species as the mammal being evaluated. In some cases, control serum can be obtained from one or more mammals that are from the same species as the mammal being evaluated. When diagnosing SLE, control serum can be isolated from healthy mammals such as healthy humans who do not have SLE. Any number of control mammals can be used to obtain the control serum. For example, control serum can be obtained from one or more healthy mammals (e.g., at least 5, at least 10, at least 15, at least 20, or more than 20 control mammals).

Any method can be used to determine whether or not a polypeptide is present at a level that is greater than or less than the average level observed in control serum. For example, the level of a particular polypeptide can be measured using, without limitation, immuno-based assays (e.g., ELISA), western blotting, arrays for detecting polypeptides, two-dimensional gel analysis, chromatographic separation, or mass spectroscopy. Methods of using arrays for detecting polypeptides include, without limitation, those described herein. Such methods can be used to determine simultaneously the relative levels of multiple polypeptides.

This document also provides methods and materials for diagnosing a mammal (e.g., a human) as having SLE disease activity. A number of measures can typically be used to define active SLE disease. Such disease activity measures include, without limitation, the SLE Disease Activity Index (SLEDAI), the Systemic Lupus Activity Measure (SLAM), a physicians global assessment (PGA), the erythrocyte sedimentation rate (ESR), the titers of anti-dsDNA antibodies, the white blood cell (WBC) count, and the hematocrit. A mammal can be diagnosed as having active or inactive SLE disease based on one or more disease activity measures. For example, a human having a PGA≧1.5 and SLEDAI≧3 can be diagnosed as having active SLE disease. In some cases, a human having a PGA≦1 and SLEDAI≦2 can be diagnosed as having inactive SLE disease.

In some embodiments, a mammal can be diagnosed as having active SLE disease if it is determined that the mammal's serum contains one or more of the polypeptides listed in Table 2, 3, or 4 at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from control mammals. In some cases, a mammal can be diagnosed as having active SLE disease if it is determined that the mammal's serum contains one or more of the polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is greater than the average level of the same one or more polypeptides observed in control serum obtained from control mammals.

Once a mammal (e.g., a human) has been diagnosed as having active SLE disease, the mammal can be subsequently evaluated or monitored over time for an increase or a decrease in SLE disease activity. For example, a mammal can be assessed as having an increased or decreased SLE disease activity if it is determined that the mammal's serum contains one or more polypeptides listed in Table 2, 3, or 4 at a level that is greater than or less than the average level of the same one or more polypeptides observed in serum obtained previously from the same mammal. In some cases, a mammal can be assessed as having an increased or decreased SLE disease activity if it is determined that the mammal's serum contains one or more polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is greater than or less than the average level of the same one or more polypeptides observed in serum obtained previously from the same mammal. For example, a mammal can be assessed as having an increased SLE disease activity if it is determined that the mammal's serum contains one or more polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is greater than the average level of the same one or more polypeptides observed in serum obtained previously from the same mammal. In some cases, a mammal can be assessed as having a decreased SLE disease activity if it is determined that the mammal's serum contains one or more polypeptides selected from the group consisting of CCL2, CCL3, CCL8, CCL19, CXCL9, CXCL10, and CXCL11 polypeptides at a level that is less than the average level of the same one or more polypeptides observed in serum obtained previously from the same mammal. A mammal can be monitored for SLE disease activity over any period of time with any frequency. For example, a mammal can be monitored every three months for one year or once a year for as long as the mammal is alive. In some cases, the SLE disease activity of a mammal can be monitored with a single follow-up assessment.

A mammal can also be assessed for SLE disease activity before, during, and after being treated for SLE. For example, a mammal can be assessed for SLE disease activity while being treated with anti-interferon therapy, hydroxychloroquinone, steroids, or immunosuppressive drugs. Assessing a mammal for SLE disease activity during treatment of the mammal for SLE can allow the effectiveness of the SLE therapy to be determined. For example, a decrease in SLE activity during or after treatment with an SLE therapy compared to the SLE activity before treatment with an SLE therapy can indicate that the SLE therapy is effective. Assessing a mammal for SLE disease activity during treatment of the mammal for SLE can also allow responders to the SLE therapy to be identified. For example, a decrease in SLE activity in a mammal during treatment with an SLE therapy compared to the SLE activity in the mammal before treatment with the SLE therapy can indicate that the mammal is a responder to the SLE therapy.

This document also provides methods and materials for identifying mammals (e.g., humans) having SLE that are likely to experience SLE disease activity. For example, future SLE disease activity in a mammal can be predicted by determining whether or not the mammal's serum contains one or more of the polypeptides listed in Table 2, 3, or 4 at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from control mammals.

This document also provides methods and materials for identifying mammals (e.g., humans) likely to respond to an anti-IFN SLE treatment. For example, the methods and materials provided herein can be used to identify SLE patients having serum containing one or more IFN-regulated chemokine polypeptides at a level that is greater than or less than the average level of the same one or more polypeptides observed in control serum obtained from control mammals. Once identified, patients can be treated with an anti-IFN treatment such as humanized anti-IFN antibodies. In some cases, the effectiveness of the anti-IFN SLE treatment can be assessed as described herein.

This document also provides arrays for detecting polypeptides. The arrays provided herein can be two-dimensional arrays, and can contain at least two different polypeptides capable of detecting polypeptides, such as antibodies (e.g., at least three, at least five, at least ten, at least 20, at least 30, at least 50, at least 100, or at least 200 different polypeptides capable of detecting polypeptides). The arrays provided herein also can contain multiple copies of each of many different polypeptides. In addition, the arrays for detecting polypeptides provided herein can contain polypeptides attached to any suitable surface (e.g., plastic or glass).

A polypeptide capable of detecting a polypeptide can be naturally occurring, recombinant, or synthetic. The polypeptides immobilized on an array also can be antibodies or antibody fragments, such as Fab′ fragments, Fab fragments, single-chain Fvs, antigen-specific polyclonal antibodies, or full-length monoclonal antibodies. Such an antibody or antibody fragment can be capable of binding specifically to a polypeptide listed in Table 2, 3, or 4. The polypeptides immobilized on the array can be members of a family such as a receptor family, ligand family, or enzyme family.

The production of monoclonal antibodies against a polypeptide target is routine using standard hybridoma technology. In addition, numerous monoclonal antibodies are available commercially. An antibody fragment can be produced by any means. For example, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody. An antibody fragment also can be produced synthetically or recombinantly from a gene encoding the partial antibody sequence. The antibody fragment can be a single chain antibody fragment. Alternatively, the fragment can include multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex.

Any method can be used to make an array for detecting polypeptides. For example, methods disclosed in U.S. Pat. No. 6,630,358 can be used to make arrays for detecting polypeptides. Arrays for detecting polypeptides can also be obtained commercially, such as from Panomics, Redwood City, Calif.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1

Identifying Polypeptides That Can Be Used as Biomarkers for SLE

SLE patients were enrolled from the Hopkins Lupus Cohort (Petri, Rheum Dis Clin North Am 26:199-213 (2000)). Healthy age- and gender-matched controls were recruited. Following informed consent, blood samples were collected with the PAXgene system (Qiagen/Becton-Dickinson, Hombrechtikon, Switzerland and Franklin Lakes, N.J.). These samples were used to generate whole blood gene expression profiles. cRNA probes were prepared from RNA purified using the PAXgene system. The cRNA probes were hybridized with Affymetrix U133A arrays (Affymetrix, Santa Clara, Calif.) using standard protocols. The levels of 82 IFN-regulated genes that distinguished 81 SLE patients from 42 healthy controls were normalized and used to assign a gene expression “score,” the IFN gene score, as described previously (Baechler et al., Proc Nat Acad Sci USA 100:2610-2615 (2003)).

Fifteen patients with high levels of IFN-regulated transcripts (IFN-hi), 15 patients with lower levels of the same transcripts (IFN-lo), and 15 matched controls were studied further. Clinical and demographic features of the two groups are compared in Table 1. The IFN-hi group was enriched for African-American women (8/15) compared with the IFN-lo group (2/15; P=0.05), and, on average, fulfilled more criteria for SLE (7.3) than IFN-lo cases (5.7; P=0.004). All of the IFN-hi cases had a history of positive anti-dsDNA Abs and either low C3 or C4 complement levels, while these features were found in only about half of the IFN-lo group. The IFN-hi group also showed evidence of more active disease than the IFN-lo group as determined by higher SLEDAI and SLAM-R scores, together with several laboratory measures characteristic of active disease (low complement C3, and elevated ESR and anti-dsDNA antibodies) at the time of the visit. There were no significant differences in medication profiles between the groups. Thus, disease severity appeared to be increased in IFN-hi as compared to IFN-lo patients.

TABLE 1
Clinical and demographic features of SLE cases
	IFN-hi	IFN-lo
	(N = 15)	(N = 15)	P
	Age (avg.)	37.7a	40.9
	Female	15b	13
	Caucasian	6	13	0.01
	African-American	8	2	0.05
	Historical
	ACR criteria (avg. #)	7.3	5.7	0.004
	Malar Rash	11	7
	Discoid Rash	3	1
	Photosensitivity	8	6
	Oral/Nasal Ulcers	8	9
	Arthritis	12	12
	Pleurisy/Pericarditis	13	7	0.03
	Renal	9	8
	Neurologic	3	0
	Hematologic	14	12
	Immunologic	15	8	0.003
	Positive ANA	15	15
	Hx low C3	15	7	0.001
	Hx low C4	14	6	0.003
	Hx anti-dsDNA Abs	15	8	0.003
	Hx anti-Ro Abs	8	3
	Hx anti-La Abs	4	0	0.05
	Current Visit
	PGA (0-3 scale)	1.5	1.1
	SLEDAI	5.6	2.3	0.008
	BILAG	4.8	3.9
	SLAM-R	5.9	2.8	0.0001
	HCT	37.3	36.9
	WBC count	5.3	7.4
	ESR	51.3	19.7	0.008
	Complement C3	80.5	109.6	0.008
	Complement C4	13.9	18.6
	Positive anti-DNA Abs	8	1	0.007
	Platelet count	264.4	181.7
	Lymphocyte count	0.8	1.6	0.013
	Prednisone (avg. dose	13 (14.6)	9 (21.9)
	mgs)
	IV steroids	6	4
	Immunosuppressives	4	7
	Steroids and/or	14	11
	immunosuppressives
	Plaquenil	12	12
	NSAID	6	4
	aContinuous variables were compared using unpaired T tests and results reported with one decimal place.
	bData indicate the number of individuals positive for the feature within each group of 15. These variables were compared using Fisher's Exact Test.

The IFN gene expression scores of the 45 study subjects are plotted in FIG. 1. Detailed clinical data were available for each visit, including the SLEDAI and SLAM disease activity measures, laboratory test results, and medication profiles. The SLEDAI (Bombardier et al., Arthritis Rheum 35:630-640 (1992)) consists of 22 defined weighted items grouped into nine organ systems. The index was calculated by summing all weighted items that were present within the previous ten days. Possible SLEDAI scores ranged from 0 to 101. The Systemic Lupus Activity Measure-Revised (SLAM-R) listed 33 clinical and laboratory manifestations of SLE (Liang et al., Arthritis Rheum 32:1107-1118 (1989)). Each manifestation was graded according to the severity of activity within the month before evaluation. Possible total scores vary from 0 to 86.

Serum was isolated from patients and control subjects by peripheral venipuncture using serum-separator vacutainer tubes (Becton-Dickinson, Franklin Lakes, N.J.). A protease inhibitor (aprotinin, 1 μg/mL) was added to each sample, and aliquots were immediately frozen at −80° C. The levels of 160 serum polypeptide analytes were measured in serum aliquots (100 μL) using custom dual-antibody sandwich immunoassay arrays, as described elsewhere (Shao et al., J Biomed Biotechnol 2003:299-307 (2003); Perlee et at., Proteome Sci 2:9 (2004)). Briefly, monoclonal capture antibodies specific for each analyte were fixed to glass slides, with 12 replicate spots for each analyte. The slides were incubated with serum samples for two hours. Each serum sample was tested in duplicate. Slides were washed and incubated with secondary biotinylated polyclonal antibodies. The signals were amplified using a “rolling circle” method (Perlee et al., Proteome Sci 2:9 (2004)).

Quality control measures included optimization of antibody pairs, use of internal controls to minimize array-to-array variation, and standardized procedures for array manufacturing (Shao et al., J Biomed Biotechnol 2003:299-307 (2003); Perlee et al., Proteome Sci 2:9 (2004)). Arrays were scanned using a Tecan LS200 scanner (Tecan, Männedorf, Switzerland), Mean Fluorescence Intensities (MFIs) were generated using customized software. MFIs were converted to concentration values using best-fit equations generated for each analyte using 15 serial dilutions of a known concentration of recombinant analyte. Four anchor-point control dilutions of recombinant analytes were included on each slide. The upper and lower limits of quantitation were defined to ensure a dynamic working range. The results are presented in Table 2. CXCL10 (IP-10) levels were measured using Luminex assays since an antibody directed against this analyte was not included on the antibody array. In addition to the 161 serum polypeptide analytes listed in Table 2, the following twelve analytes were measured using the antibody arrays: ANG, CNTF, D-DIMER DD5, D-DIMER DD6, IGFBP-3, IGFBP-6, IL-17, MMP9, PROTEIN C, PROTEIN S, TIMP-2, and VAP-1. These 12 analytes were excluded from further analysis because the concentrations present in ≧80% of the samples were at the upper or lower limits of detection.

TABLE 2
Mean Serum Analyte Concentrations (pg/ml) for 15 IFN-hi SLE Cases, 15 IFN-lo SLE Cases, and 15 Controls
								All SLE v.	IFN-hi v.	IFN-lo v.	IFN-hi v.
	Common	Accession	Gene					Ctrl	Ctrl	Ctrl	IFN-lo
	Analyte	name	No.	ID	All SLE Avg	IFN-hi Avg	IFN-lo Avg	Ctrl Avg	FC	p-val	FC	p-val	FC	p-val	FC	p-val
1	ACE	angiotensin I	NP_000780.1	1636	39757a	34268	45246	53205	−1.34	9.1E−03	−1.55	8.0E−04	−1.18	1.9E−01	−1.32	5.6E−02
		converting
		enzyme 1
2	ACE2	angiotensin I	NP_068576.1	59272	2639	1968	3310	2871	−1.09	5.6E−01	−1.46	4.0E−03	1.15	4.8E−01	−1.68	3.4E−02
		converting
		enzyme 2
3	BDNF	brain-derived	NP_001700.2	627	928	1182	673	764	1.21	2.1E−01	1.55	3.7E−02	−1.14	4.0E−01	1.76	1.4E−02
		neurotrophic
		factor
4	BLC	CXCL13	NP_006410.1	10563	209	233	185	107	1.96	8.8E−04	2.18	3.4E−03	1.73	7.5E−02	1.26	3.7E−01
5	EGF	epidermal	NP_001954.1	1950	61	62	59	29	2.12	1.8E−03	2.16	7.5E−03	2.08	1.0E−02	1.04	8.6E−01
		growth factor
6	FGFR3	fibroblast	NP_000133.1	2261	632	625	639	934	−1.48	9.3E−03	−1.50	1.0E−02	−1.46	1.7E−02	−1.02	8.6E−01
	(IIIC)	growth factor
		receptor 3
		(IIIC)
7	FGF2	fibroblast	NP_001997.4	2247	544	575	513	768	−1.41	4.5E−04	−1.34	2.2E−02	−1.50	1.1E−04	1.12	4.1E−01
		growth factor 2
8	GDNF	glial cell	NP_000505.1	2668	9	10	8	6	1.43	3.9E−04	1.53	9.2E−03	1.33	1.2E−02	1.15	3.3E−01
		derived
		neurotrophic
		factor
9	GROB	CXCL2	NP_002080.1	2920	936	1091	780	513	1.82	1.7E−02	2.13	3.2E−02	1.52	2.5E−01	1.40	3.5E−01
10	ICAM3	intercellular	NP_002153.1	3385	9702	11519	7884	7394	1.31	8.4E−03	1.56	3.7E−03	1.07	5.6E−01	1.46	1.6E−02
		adhesion
		molecule 3
11	IFNW	interferon	NP_002168.1	3467	7548	7576	7519	14195	−1.88	7.0E−04	−1.87	9.7E−04	−1.89	7.4E−04	1.01	9.6E−01
		omega
12	IL15	interleukin 15	NP_000576.1	3600	433	515	351	274	1.58	8.7E−03	1.88	4.3E−02	1.28	9.0E−04	1.47	1.6E−01
13	IL18	interleukin 18	NP_001553.1	3606	129	151	108	88	1.46	4.2E−03	1.71	1.5E−03	1.22	2.5E−01	1.40	4.8E−02
14	IL2SRA	interleukin 2	NP_000408.1	3559	890	940	840	521	1.71	3.1E−05	1.81	1.1E−03	1.61	1.0E−03	1.12	4.1E−01
		receptor,
		alpha
15	IL5	interleukin 5	NP_000870.1	3567	3	4	3	2	1.54	5.1E−04	1.72	7.4E−03	1.37	1.0E−02	1.26	1.8E−01
16	IL6	interleukin 6	NP_000591.1	3569	10	11	9	3	2.83	9.1E−07	3.11	7.9E−05	2.55	3.2E−03	1.22	3.5E−01
17	IL8	interleukin 8	NP_000575.1	3576	8	9	8	4	2.31	8.6E−05	2.49	4.5E−03	2.14	4.8E−03	1.16	5.3E−01
18	IP10b	CXCL10	NP_001556.1	3627	77	120	23	13	5.82	1.4E−03	9.09	1.5E−03	1.74	2.6E−02	5.82	3.1E−03
19	ITAC	CXCL11	NP_005400.1	6373	204	263	145	167	1.23	1.5E−01	1.58	4.2E−02	−1.15	2.1E−01	1.81	1.8E−02
20	MCP1	CCL2	NP_002973.1	6347	92	135	50	24	3.78	8.5E−04	5.52	3.1E−03	2.04	5.7E−02	2.71	1.9E−02
21	MCP2	CCL8	NP_005614.2	6355	38	51	26	15	2.48	9.1E−05	3.27	6.9E−04	1.69	2.2E−02	1.93	1.4E−02
22	MCP3	CCL7	NP_006264.2	6354	48	50	46	31	1.57	9.6E−06	1.63	2.1E−04	1.51	1.5E−03	1.08	5.0E−01
23	MIG	CXCL9	NP_002407.1	4283	127	180	74	56	2.29	1.1E−02	3.24	1.4E−02	1.34	3.5E−01	2.42	3.9E−02
24	MIP1A	CCL3		6348	370	438	301	219	1.69	4.9E−07	2.00	1.8E−06	1.38	1.1E−03	1.45	6.5E−04
25	MIP3A	CCL20	NP_004582.1	6364	79	81	76	117	−1.48	6.0E−03	−1.43	1.1E−02	−1.53	4.1E−03	1.06	4.0E−01
26	MIP3B	CCL19	NP_006265.1	6363	158	218	98	77	205	1.6E−04	2.82	9.0E−05	1.27	1.4E−01	2.22	5.1E−04
27	MMP7	matrix	NP_002414.1	4316	1252	1295	1209	790	1.58	2.1E−03	1.64	6.4E−03	1.53	8.0E−02	1.07	7.5E−01
		metalloproteinase 7
28	PDGFRA	platelet-	NP_006197.1	5156	23663	24381	22945	35538	−1.50	7.2E−04	−1.46	2.4E−03	−1.55	5.4E−04	1.06	5.6E−01
		derived
		growth factor
		receptor,
		alpha
29	TARC	CCL17	NP_002978.1	6361	68	74	63	42	1.61	2.3E−02	1.74	3.1E−02	1.48	2.4E−01	1.17	6.0E−01
30	TGFBRIII	transforming	NP_003234.2	7049	16431	17611	15250	7541	2.18	1.8E−03	2.34	5.2E−03	2.02	2.6E−02	1.15	5.4E−01
		growth factor,
		beta receptor
		III
31	41BB	TNFR	NP_001552.2	3604	208	195	221	235	−1.13	3.7E−01	−1.20	1.8E−0.1	−1.06	7.3E−01	−1.13	4.8E−01
		Superfamily,
		member 9
32	6CKINE	CCL21	NP_002980.1	6366	845	907	782	642	1.32	8.3E−05	1.41	2.8E−04	1.22	5.4E−03	1.16	5.8E−02
33	AFP	alpha-	NP_001125.1	174	769	1089	448	496	1.55	4.1E−01	2.19	3.7E−01	−1.11	5.4E−01	2.43	3.4E−01
		fetoprotein
34	AGRP	agouti related	NP_001129.1	181	213	216	211	254	−1.19	3.0E−02	−1.17	7.0E−02	−1.20	4.2E−02	1.03	7.7E−01
		protein
		homolog
		(mouse)
35	ALCAM	activated	NP_001618.1	214	13364	14278	12450	15481	−1.16	7.6E−03	−1.08	2.2E−01	−1.24	1.2E−03	1.15	8.6E−02
		leukocyte cell
		adhesion
		molecule
36	AR	amphiregulin	NP_001648.1	374	35	37	32	36	−1.05	5.2E−01	1.01	9.2E−01	−1.12	1.5E−01	1.13	1.5E−01
37	BNGF	nerve growth	NP_002497.1	4803	1109	1156	1062	1234	−1.11	1.6E−01	−1.07	5.0E−01	−1.16	6.8E−02	1.09	4.0E−01
		factor, beta
		protein
38	BTC	betacellulin	NP_001720.1	685	703	772	633	761	−1.08	5.3E−01	1.01	9.4E−01	−1.20	1.3E−01	1.22	3.8E−01
39	CA125	mucin 16		94025	530	505	555	718	−1.35	7.9E−02	−1.42	5.2E−02	−1.29	1.7E−01	−1.10	5.3E−01
40	CD141	thrombomodulin	NP_000352.1	7056	15308	14839	15776	18667	−1.22	6.2E−02	−1.26	9.1E−02	−1.18	1.7E−01	−1.06	6.9E−01
41	CD27	TNFR	NP_001233.1	939	5116	5752	4480	4958	1.03	6.5E−01	1.16	1.7E−01	−1.11	1.3E−01	1.28	4.4E−02
		superfamily,
		member 7
42	CD30	TNFR	NP_001234.1	943	4991	4971	5010	5873	−1.18	2.7E−02	−1.18	1.1E−01	−1.17	5.1E−02	−1.01	9.5E−01
		superfamily,
		member 8
43	CD40	TNFR	NP_001241.1	958	330	328	332	345	−1.05	6.3E−01	−1.05	6.3E−01	−1.04	7.2E−01	−1.01	9.0E−01
		superfamily
		member 5
44	CD44V6	CD44 antigen	NP_000601.3	960	14912	14651	15173	17100	−1.15	2.5E−01	−1.17	1.9E−01	−1.13	3.3E−01	−1.04	4.8E−01
45	CNTF RA	ciliary		12803	253	244	262	302	−1.19	1.9E−03	−1.24	9.2E−04	−1.15	7.8E−02	−1.07	4.4E−01
		neurotrophic
		factor
46	CRP	c-reactive	NP_000558.2	1401	10184	10229	10139	10850	−1.07	2.3E−01	−1.06	3.7E−01	−1.07	2.8E−01	1.01	9.0E−01
		protein
47	CTACK	CCL27	NP_006655.1	10850	718	692	744	901	−1.25	7.7E−03	−1.30	4.0E−03	−1.21	7.1E−02	−1.07	5.2E−01
48	DR6	TNFR	NP_055267.1	27242	2831	2977	2684	2508	1.13	1.8E−01	1.19	2.1E−01	1.07	4.7E−01	1.11	4.6E−01
		superfamily,
		member 21
49	ENA78	CXCL5	NP_002985.1	6374	3035	3974	2096	2470	1.23	4.2E−01	1.61	1.6E−01	−1.18	6.3E−01	1.90	1.3E−01
50	ENDOSTATIN	procollagen,	NP_085059.1	80781	8586	8193	8978	9916	−1.15	7.1E−02	−1.21	5.2E−02	−1.10	2.9E−01	−1.10	4.2E−01
		type XVIII,
		alpha 1
51	EOT	CCL11	NP_002977.1	6356	142	142	142	114	1.25	1.1E−01	1.25	1.7E−01	1.25	2.3E−01	−1.00	1.0E+00
52	EOT2	CCL24	NP_002982.2	6369	335	229	441	488	−1.46	3.2E−01	−2.31	9.2E−02	−1.11	7.9E−01	−1.92	6.3E−02
53	EOT3	CCL26	NP_006063.1	10344	590	612	568	745	−1.26	7.0E−04	−1.22	2.1E−02	−1.31	1.1E−03	1.08	4.6E−01
54	ERBB1	epidermal	NP_005219.2	1956	15201	13979	16423	18595	−1.22	9.1E−03	−1.33	4.4E−03	−1.13	1.5E−01	−1.17	1.5E−01
		growth factor
		receptor
55	ERBB2	v-erb-b2	NP_001005862.1	2064	3910	3647	4173	4905	−1.25	1.1E−03	−1.34	2.5E−04	−1.18	2.3E−02	−1.14	7.0E−02
		erythroblastic
		leukemia
		homolog 2
56	ESELECTIN	selectin E	NP_000441.1	6401	5889	6567	5210	6034	−1.02	8.5E−01	1.09	5.9E−01	−1.16	2.9E−01	1.26	1.1E−01
57	ET3	endothelin	NP_000105.1	1908	2414	2455	2373	2867	−1.19	1.9E−01	−1.17	2.5E−01	−1.21	1.8E−01	1.03	7.6E−01
58	FAS	TNFR	NP_000034.1	355	558	596	520	1734	−3.11	3.7E−01	−2.91	3.8E−01	−3.34	3.5E−01	1.15	2.7E−01
		superfamily,
		member 6
59	FASL	fas ligand	NP_000630.1	356	1975	2032	1918	2542	−1.29	8.3E−02	−1.25	1.6E−01	−1.33	6.6E−02	1.06	6.6E−01
60	FGF1	fibroblast	NP_000791.1	2246	91	90	91	89	1.02	8.6E−01	1.01	9.3E−01	1.02	8.6E−01	−1.01	9.2E−01
		growth factor 1
61	FGF4	fibroblast	NP_001998.1	2249	815	824	806	1117	−1.37	1.3E−03	−1.36	4.6E−03	−1.39	1.6E−03	1.02	8.2E−01
		growth factor 4
62	FGF6	fibroblast	NP_066276.2	2251	518	503	532	619	−1.20	7.7E−02	−1.23	9.3E−02	−1.16	1.6E−01	−1.06	6.6E−01
		growth factor 6
63	FGF7	fibroblast	NP_002000.1	2252	221	232	211	231	−1.40	7.1E−01	1.00	9.8E−01	−1.09	4.6E−01	1.10	3.4E−01
		growth factor 7
64	FGFR3(IIIB)	fibroblast	NP_000133.1	2261	865	860	871	1011	−1.17	1.2E−01	−1.18	1.4E−01	−1.16	1.8E−01	−1.01	9.1E−01
		growth factor
		receptor 3
		(IIIB)
65	FLT3LIG	fms-related	NP_001450.2	2323	132	157	107	121	1.09	4.5E−01	1.30	1.1E−01	−1.13	2.7E−01	1.47	3.7E−02
		tyrosine
		kinase 3
		ligand
66	FOLLISTATIN	follistatin	NP_006341.1	10468	1958	1547	2369	1696	1.15	5.4E−01	−1.10	2.6E−01	1.40	4.2E−01	−1.53	3.3E−01
67	FRACTALKINE	CX3CL1	NP_002987.1	6376	752	688	816	852	−1.13	4.0E−01	−1.24	1.4E−01	−1.04	8.4E−01	−1.19	4.4E−01
68	GCP2	CXCL6	NP_002984.1	6372	253	325	180	207	1.22	3.8E−01	1.57	2.1E−01	−1.15	5.2E−01	1.81	1.5E−01
69	GCSF	colony	NP_000750.1	1440	1220	1235	1205	1252	−1.03	7.9E−01	−1.01	9.1E−01	−1.04	7.3E−01	1.02	8.4E−01
		stimulating
		factor 3
70	GMCSF	colony	NP_000749.2	1437	27	30	24	23	1.19	1.1E−01	1.32	4.0E−02	1.05	6.5E−01	1.26	7.2E−02
		stimulating
		factor 2
71	GROG	CXCL3	NP_002081.2	2921	901	1195	607	741	1.22	3.3E−01	1.61	1.5E−01	−1.22	5.5E−02	1.97	6.7E−02
72	HBEGF	heparin-	NP_001936.1	1839	94	90	98	104	−1.11	2.4E−01	−1.16	1.8E−01	−1.06	5.6E−01	−1.10	4.6E−01
		binding EGF-
		like growth
		factor
73	HCC1	RNA-binding	NP_004893.1	9584	5713	5560	5865	5514	1.04	3.6E−01	1.01	8.5E−01	1.06	2.3E−01	−1.05	3.4E−01
		region
		containing 2
74	HCC4	CCL16	NP_004581.1	6360	4651	4921	4381	4758	−1.02	8.2E−01	1.03	7.7E−01	−1.09	4.7E−01	1.12	3.6E−01
75	HCG	human	NP_000726.1	1081	1491	2364	619	745	2.00	4.0E−01	3.17	3.7E−01	−1.20	2.9E−02	3.82	3.3E−01
		chorionic
		gonadotropin
76	HGF	hepatocyte	NP_000592.3	3082	919	841	997	923	−1.00	9.8E−01	−1.10	5.9E−01	1.08	6.5E−01	−1.19	3.9E−01
		growth factor
77	HSP70	heat shock	NP_005336.2	3303	16077	16000	16154	19234	−1.20	1.4E−01	−1.20	1.6E−01	−1.19	1.8E−01	−1.01	9.3E−01
		protein 70
78	HVEM	TNFR	NP_003811.2	8764	1938	1938	1938	1696	1.14	2.0E−01	1.14	3.8E−01	1.14	2.8E−01	1.00	1.0E+00
		superfamily,
		member 14
79	I309	CCL1	NP_002972.1	6346	26	29	24	29	−1.10	3.7E−01	−100	9.8E−01	−1.23	9.8E−02	1.22	4.7E−02
80	ICAM1	intercellular	NP_000192.1	3383	8461	8729	8193	9319	−1.10	6.0E−01	−1.07	7.5E−01	−1.14	5.1E−01	1.07	7.2E−01
		adhesion
		molecule 1
81	lFNA	interferon	NP_076918.1	3439	29	24	34	27	1.10	6.8E−01	−1.10	6.4E−01	1.29	4.6E−01	1.41	3.3E−01
		alpha
82	IFNG	interferon,		3458	62	62	61	74	−1.21	7.6E−02	−1.21	1.3E−01	−1.21	1.2E−01	1.00	9.8E−01
		gamma
83	IGFBP1	insulin-like	NP_000587.1	3484	33784	39396	28171	34702	−1.03	9.3E−01	1.14	7.0E−01	−1.23	5.7E−01	1.40	3.2E−01
		growth factor
		binding
		protein 1
84	IGFBP2	insulin-like	NP_000588.2	3485	50266	51042	49490	48658	1.03	5.7E−01	1.05	4.3E−01	1.02	8.1E−01	1.03	6.3E−01
		growth factor
		binding
		protein 2
85	IGFBF4	insulin-like	NP_001543.1	3487	13884	13535	14233	15258	−1.10	1.7E−01	−1.13	1.4E−01	−1.07	4.0E−01	−1.05	5.9E−01
		growth factor
		binding
		protein 4
86	IGFII	insulin-like	NP_000603.1	3481	2449	2704	2193	3050	−1.25	7.2E−02	−1.13	4.3E−01	−1.39	1.1E−02	1.23	2.0E−01
		growth factor 2
87	IGFIR	insulin-like	NP_000866.1	3480	947	903	991	1278	−1.35	3.4E−02	−1.41	3.6E−02	−1.29	7.7E−02	−1.10	5.4E−01
		growth factor
		1 receptor
88	IL10RB	interleukin 10	NP_000619.3	3588	226	226	225	255	−1.13	2.1E−02	−1.13	1.5E−01	−1.13	2.9E−02	1.00	9.7E−01
		receptor, beta
89	IL12P40	interlukin	NP_002178.2	3593	1345	1389	1300	1577	−1.17	1.0E−01	−1.13	2.2E−01	−1.21	1.1E−01	1.07	5.7E−01
		12B
90	IL13	interleukin 13	NP_002179.2	3596	35	36	34	32	1.08	4.2E−01	1.10	4.0E−01	1.05	6.4E−01	1.05	6.5E−01
91	IL16	interleukin 16	NP_004504.3	3603	1541	1538	1545	1670	−1.08	1.9E−01	−1.09	3.0E−01	−1.08	3.2E−01	−1.00	9.6E−01
92	IL1A	interleukin 1,	NP_000566.3	3552	7	7	6	6	1.04	6.4E−01	1.10	2.5E−01	−1.03	7.6E−01	1.13	9.7E−01
		alpha
93	IL1B	interleukin 1,	NP_000567.1	3553	6	7	6	6	1.09	3.1E−01	1.16	1.2E−01	1.01	9.1E−01	1.15	1.6E−01
		beta
94	IL1RA	interleukin 1	NP_000568.1	3557	77	81	73	56	1.39	3.5E−03	1.47	1.9E−03	1.32	1.3E−01	1.11	5.2E−01
		receptor
		antagonist
95	IL1SR1	interleukin 1	NP_000868.1	3554	4374	4598	4150	5526	−1.26	1.1E−03	−1.20	7.7E−02	−1.33	1.4E−04	1.11	3.9E−01
		receptor type 1
96	IL1SRII	interleukin 1	NP_004624.1	7850	3692	3721	3662	5262	−1.43	1.4E−04	−1.41	5.2E−04	−1.44	1.6E−04	1.02	8.5E−01
		receptor type 2
97	IL2	interleukin 2	NP_000577.2	3558	3	4	3	3	1.05	8.1E−02	1.10	8.1E−02	1.00	3.3E−01	1.10	8.1E−02
98	IL2RB	interleukin 2	NP_000869.1	3560	9845	9852	9838	11478	−1.17	4.6E−02	−1.17	1.5E−01	−1.17	7.8E−02	1.00	9.9E−01
		receptor, beta
99	IL2RG	interleukin 2	NP_000197.1	3561	436	434	439	535	−1.23	8.6E−02	−1.23	1.1E−01	−1.22	1.7E−01	−1.01	9.5E−01
		receptor,
		gamma
100	IL3	interleukin 3	NP_000579.2	3562	157	169	144	168	−1.07	5.2E−01	1.01	9.5E−01	−1.17	1.8E−01	1.17	1.8E−01
101	IL4	interleukin 4	NP_000580.1	3565	34	34	34	35	−1.04	6.3E−01	−1.04	7.2E−01	−1.04	6.2E−01	1.01	9.4E−01
102	IL5RA	interleukin 5	NP_000555.2	3568	1073	1037	1109	1318	−1.23	8.0E−02	−1.27	1.1E−01	−1.19	1.9E−01	−1.07	7.0E−01
		receptor,
		alpha
103	IL7	interleukin 7	NP_000871.1	3574	11	12	10	9	1.23	4.3E−02	1.36	2.4E−02	1.11	3.6E−01	1.22	1.2E−01
104	IL9	interleukin 9	NP_000581.1	3578	5129	5059	5199	5611	−1.09	4.5E−01	−1.11	5.0E−01	−1.08	5.9E−01	−1.03	8.8E−01
105	LEPTIN	leptin	NP_000221.1	3952	61316	61457	61176	52604	1.17	2.0E−01	1.17	2.4E−01	1.16	2.4E−01	1.00	9.6E−01
106	LIF	leukemia	NP_002300.1	3976	682	690	675	745	−1.09	3.3E−01	−1.08	4.8E−01	−1.10	3.0E−01	1.02	8.2E−01
		inhibitory
		factor
107	LIFRA	leukemia	NP_002301.1	3977	6449	6887	6012	6707	−1.04	6.6E−01	1.03	8.2E−01	−1.12	3.4E−01	1.15	3.5E−01
		inhibitory
		factor
		receptor
108	LSELECTIN	selectin L	NP_000646.1	6402	24767	25141	24393	24365	1.02	7.1E−01	1.03	5.0E−01	1.00	9.9E−01	1.03	7.0E−01
109	LTBR	TNFR	NP_002333.1	4055	518	543	493	513	1.01	9.2E−01	1.06	7.1E−01	−1.04	6.5E−01	1.10	5.5E−01
		superfamily,
		member 3
110	LYMPHOTACTIN	XCL1	NP_002986.1	6375	632	653	611	814	−1.29	5.4E−03	−1.25	2.9E−02	−1.33	3.3E−03	1.07	4.8E−01
111	MCP4	CCL13	NP_005399.1	6357	219	223	215	222	−1.01	8.8E−01	1.00	9.7E−01	−1.03	7.7E−01	1.04	7.8E−01
112	MCSF	colony	NP_000748.3	1435	138	155	122	149	−1.08	4.4E−01	1.04	7.6E−01	−1.23	5.0E−02	1.27	2.7E−02
		stimulating
		factor 1
113	MCSFR	colony	NP_005202.2	1436	53897	55653	52141	55491	−1.03	6.8E−01	1.00	9.7E−01	−1.06	5.1E−01	1.07	5.1E−01
		stimulating
		factor 1
		receptor
114	MIF	macrophage	NP_002406.1	4282	50594	46404	54784	46258	1.09	1.7E−01	1.00	9.7E−01	1.18	3.4E−02	−1.18	6.9E−02
		migration
		inhibitory
		factor
115	MIP1B	CCL4	NP_002975.1	6351	40	69	11	10	4.06	1.8E−01	7.02	1.9E−01	1.10	6.5E−01	6.36	1.9E−01
116	MIP1D	CCL15		6359	922	943	901	1006	−1.09	6.6E−01	−1.07	7.8E−01	−1.12	6.5E−01	1.05	8.6E−01
117	MMP1	matrix	NP_002412.1	4312	1645	1780	1510	1744	−1.06	6.5E−01	1.02	8.9E−01	−1.15	4.1E−01	1.18	4.0E−01
		metalloproteinase 1
118	MMP10	matrix	NP_002416.1	4319	1828	1994	1662	1728	1.06	4.0E−01	1.15	1.5E−01	−1.04	5.5E−01	1.20	9.3E−02
		metalloproteinase
		10
119	MMP2	matrix	NP_004521.1	4313	24751	24283	25187	25324	−1.02	8.0E−01	−1.04	6.9E−01	−1.01	9.7E−01	−1.04	8.2E−01
		metalloproteinase 2
120	MMP8	matrix	NP_002415.1	4317	17360	14133	20587	19345	−1.11	4.5E−01	−1.37	9.6E−02	1.06	7.0E−01	−1.46	7.8E−02
		metalloproteinase 8
121	MPIF1	CCL23	NP_005055.2	6368	511	484	538	412	1.24	6.2E−02	1.17	3.7E−01	1.31	2.7E−02	−1.11	5.4E−01
122	NAP2	CXCL7	NP_002695.1	5473	9917	9888	9947	10197	−1.03	3.9E−01	−1.03	4.6E−01	−1.03	5.1E−01	−1.01	9.0E−01
123	NEUTELAST	elastase 2,	NP_001963.1	1991	16767	15699	17836	15248	1.10	2.6E−01	1.03	7.8E−01	1.17	1.6E−01	−1.14	3.1E−01
		neutrophil
124	NT3	neurotrophin 3	NP_002518.1	4908	128	135	121	151	−1.18	9.7E−02	−1.12	2.8E−01	−1.25	3.0E−02	1.12	5.8E−02
125	NT4	neurotrophin 4	NP_006170.1	4909	124	141	108	147	−1.19	1.9E−01	−1.05	7.4E−01	−1.37	2.7E−02	1.31	1.9E−02
126	OPN	osteopontin	NP_000573.1	6696	6685	6704	6666	5352	1.25	4.9E−02	1.25	1.4E−01	1.25	8.3E−02	1.01	9.7E−01
127	OSM	oncostatin M	NP_065391.1	5008	21	24	18	18	1.16	1.9E−01	1.31	1.9E−01	1.00	3.3E−01	1.31	2.0E−01
128	PAI1	plasminogen	NP_000593.1	5054	8847	8961	8733	9410	−1.06	4.5E−02	−1.05	1.4E−01	−1.08	1.6E−01	1.03	6.8E−01
		activator
		inhibitor type 1
129	PAIII	serine	NP_002566.1	5055	1128	1444	811	1026	1.10	7.5E−01	1.41	5.1E−01	−1.26	1.2E−03	1.78	3.3E−01
		proteinase
		inhibitor,
		member 2
130	PARC	p53-	NP_055904.1	23113	948	1001	896	1088	−1.15	3.0E−01	−1.09	5.5E−01	−1.22	1.6E−01	1.12	2.6E−01
		associated
		parkin-like
		cytoplasmic
		protein
131	PDGFRB	platelet-	NP_002600.1	5159	948	874	1023	1211	−1.28	5.5E−02	−1.39	2.4E−02	−1.18	1.9E−01	−1.17	1.8E−01
		derived
		growth factor
		receptor, beta
132	PECAM1	platelet/endothelial	NP_000433.2	5175	7336	7764	6908	6065	1.21	1.7E−02	1.28	1.2E−02	1.14	1.9E−01	1.12	2.5E−01
		cell
		adhesion
		molecule
133	PEDF	pigment	NP_002606.3	5176	66553	63537	69570	67059	−1.01	9.0E−01	−1.06	4.1E−01	1.04	5.8E−01	−1.09	1.5E−01
		epithelium
		derived
		factor,
		member 1
134	PF4	CXCL4	NP_002610.1	5196	3072	3007	3137	3412	−1.11	5.2E−02	−1.13	4.2E−02	−1.09	2.2E−01	−1.04	5.7E−01
135	PLGF	placental	NP_002623.2	5228	37	40	34	28	1.32	2.6E−03	1.42	9.8E−04	1.22	4.6E−02	1.17	6.4E−02
		growth factor
136	PROLACTIN	prolactin	NP_000939.1	5617	13500	15277	11723	10551	1.28	1.1E−01	1.45	1.5E−01	1.11	4.8E−01	1.30	3.1E−01
137	PSELECTIN	CD62P	NP_002996.1	6403	36020	33455	38586	44403	−1.23	2.5E−01	−1.33	1.9E−01	−1.15	4.8E−01	−1.15	5.0E−01
138	RANK	TNFR	NP_003830.1	8792	245	217	273	252	−1.03	8.1E−01	−1.16	2.9E−01	1.08	6.2E−01	−1.26	2.3E−01
		superfamily,
		member 11a
139	RANTES	CCL5	NP_002976.2	6352	2101	2101	2102	1875	1.12	2.1E−02	1.12	3.0E−02	1.12	2.8E−02	−1.00	9.8E−01
140	SCF	KIT ligand	NP_000890.1	4254	98	109	87	119	−1.21	1.1E−01	−1.09	5.1E−01	−1.37	1.8E−02	1.26	5.3E−02
141	SCFR	v-kit 4 feline	NP_000213.1	3815	5973	5453	6493	7516	−1.26	1.2E−03	−1.38	6.3E−05	−1.16	9.7E−02	−1.19	9.0E−02
		sarcoma viral
		oncogene
142	SGP130	neutrophil		4827	34497	34870	34123	47921	−1.39	4.2E−05	−1.37	8.7E−04	−1.40	2.8E−04	1.02	8.4E−01
		migration
		interleukin-1
143	ST2	receptor-like 1	NP_003847.2	9173	673	743	602	750	−1.11	3.9E−01	−1.01	9.6E−01	−1.24	1.2E−01	1.23	3.2E−01
144	SURVIVIN	baculoviral	NP_001159.1	332	10889	9750	12028	13424	−1.23	1.3E−01	−1.38	3.1E−02	−1.12	5.1E−01	−1.23	2.3E−01
		IAP repeat-
		containing 5
145	TGFA	transforming	NP_003227.1	7039	168	172	165	221	−1.31	1.6E−06	−1.28	5.1E−04	−1.34	5.5E−06	1.05	5.5E−01
		growth factor,
		alpha
146	TIE2	TEK tyrosine	NP_000450.1	7010	7410	7302	7518	9238	−1.25	5.8E−02	−1.27	7.2E−02	−1.23	1.1E−01	−1.03	8.2E−01
		kinase,
		endothelial
		tissue
147	TIMP1	inhibitor of	NP_003245.1	7076	57178	60895	53461	43261	1.32	5.5E−04	1.41	1.1E−04	1.24	5.7E−02	1.14	1.8E−01
		metalloproteinase 1
148	TNFA	TNF	NP_000585.2	7124	50	57	43	42	1.19	1.7E−01	1.35	6.6E−02	1.02	8.5E−01	1.32	6.3E−02
		superfamily,
		member 2
149	TNFB	TNF	NP_000586.2	4049	113	119	106	98	1.15	7.6E−03	1.22	3.4E−02	1.09	9.5E−02	1.12	2.3E−01
		superfamily,
		member 1
150	TNFR1	TNFR	NP_001056.1	7132	268	319	217	217	1.23	2.3E−01	1.47	7.1E−02	−1.00	1.0E+00	1.47	6.5E−02
		superfamily,
		member 1A
151	TRAILR1	TNFR	NP_003835.2	8797	98	103	93	139	−1.43	3.0E−06	−1.36	1.1E−03	−1.49	3.7E−06	1.10	3.5E−01
		superfamily,
		member 10a
152	TRAILR4	TNFR	NP_003831.2	8793	3354	3719	2989	2658	1.26	3.0E−02	1.40	1.2E−02	1.12	3.6E−01	1.24	1.0E−01
		member 10d
153	TSH	thyroid	NP_000540.1	7252	375	398	353	416	−1.11	2.2E−01	−1.05	6.3E−01	−1.18	7.6E−02	1.12	1.8E−01
		stimulating
		hormone
154	UPA	plasminogen	NP_002649.1	5328	409	384	435	384	1.07	2.7E−01	1.00	9.9E−01	1.13	9.5E−02	−1.13	1.5E−01
		activator,
		urokinase
155	UPAR	plasminogen	NP_001005376.1	5329	4047	3833	4260	3729	1.09	1.7E−01	1.03	6.9E−01	1.14	7.8E−02	−1.11	1.9E−01
		activator,
		urokinase
		receptor
156	VCAM1	vascular cell	NP_001069.1	7412	43378	47459	39298	36307	1.19	1.2E−01	1.31	5.9E−02	1.08	5.1E−01	1.21	1.4E−01
		adhesion
		molecule 1
157	VECADHERIN	cadherin 5,	NP_001786.1	1003	31057	31538	30575	29957	1.04	5.3E−01	1.05	5.1E−01	1.02	7.7E−01	1.03	7.3E−01
		type 2, VE-
		cadherin
158	VEGF	vascular	NP_003367.3	7422	421	494	347	357	1.18	2.2E−01	1.38	1.8E−01	−1.03	5.3E−01	1.42	1.5E−01
		endothelial
		growth factor
159	VEGFD	vascular	NP_004460.1	2277	7109	7107	7112	7608	−1.07	2.7E−01	−1.07	4.1E−01	−1.07	3.3E−01	−1.00	9.9E−01
		endothelial
		growth factor D
160	VEGFR2	kinase insert	NP_002244.1	3791	2714	2653	2775	2813	−1.04	7.0E−01	−1.06	6.0E−01	−1.01	9.2E−01	−1.05	7.7E−01
		domain
		receptor
161	VEGFR3	fms-related	NP_002011.1	2324	1461	1389	1533	1440	1.02	8.5E−01	−1.04	6.7E−01	1.07	5.5E−01	−1.10	3.6E−01
		tyrosine
		kinase 4
apg/ml
bIP-10 was measured by Luminex bead immunoassays

Of the 161 analytes measured, the serum levels of 30 (19%) analytes were significantly different in at least one inter-group comparison (mean fold change (FC)≧1.5 and p<0.05 by the Student's unpaired t-test). The data are presented in Table 3 as mean fold-change of the group comparisons. Serum levels of all 30 analytes differed between the IFN-hi SLE group and the control group. Serum levels of most analytes varied between the IFN-lo SLE group and the control group, or between all SLE cases and controls. The majority of analytes identified (23/30, 77%) exhibited higher serum levels in one or more of the SLE groups compared to controls. The levels of most CD4+ T helper-1 (TH1) cytokine polypeptides (e.g., IL-2, TNF-α, and IFN-γ) and TH2 cytokine polypeptides (e.g., IL-4, IL-9, IL-10, and IL-13) were similar in both the SLE and control groups (Table 2).

TABLE 3
Thirty polypeptide analytes dysregulated in SLE serum
	All
	SLE v Ctrl	SLE IFN-hi v	SLE IFN-lo v	SLE IFN-hi v
Analyte	FC	Ctrl FC	Ctrl FC	IFN-lo FC
IFN-ω	−1.88***	−1.87***	−1.89***	1.01
ACE	−1.34**	−1.55***	−1.18	−1.32
FGF RIII	−1.48**	−1.50*	−1.46*	−1.02
ACE-2	−1.09	−1.46**	1.15	−1.68*
PDGF-	−1.50***	−1.46**	−1.55***	1.06
RA
CCL20	−1.48**	−1.43*	−1.53**	1.06
(MIP-3A)
FGF-2	−1.41***	−1.34*	−1.50***	1.12
GDNF	1.43***	1.53**	1.33*	1.15
BDNF	1.21	1.55*	−1.14	1.76*
ICAM3	1.31**	1.56**	1.07	1.46*
CXCL11	1.23	1.58*	−1.15	1.81*
(I-TAC)
CCL7	1.57*****	1.63***	1.51**	1.08
(MCP-3)
MMP7	1.58**	1.64**	1.53	1.07
IL-18	1.46**	1.71**	1.22	1.40*
IL-5	1.54***	1.72**	1.37*	1.26
CCL17	1.61*	1.74*	1.48	1.17
(TARC)
IL-2SRA	1.71****	1.81**	1.61**	1.12
IL-15	1.58**	1.88*	1.28***	1.47
CCL3	1.69*****	2.00*****	1.38**	1.45***
(MIP-1A)
CXCL2	1.82*	2.13*	1.52	1.40
(GROB)
EGF	2.12**	2.16**	2.08*	1.04
CXCL13	1.96***	2.18**	1.73	1.26
(BLC)
TGF-B	2.18**	2.34**	2.02*	1.15
RIII
CXCL8	2.31****	2.49**	2.14**	1.16
(IL-8)
CCL19	2.05***	2.82****	1.27	2.22***
(MIP-3B)
IL-6	2.83*****	3.11****	2.55**	1.22
CXCL9	2.29*	3.24*	1.34	2.42*
(MIG)
CCL8	2.48****	3.27***	1.69*	1.93*
(MCP-2)
CCL2	3.78***	5.52**	2.04	2.71*
(MCP-1)
CXCL10	5.82**	9.09**	1.74*	5.82**
(IP-10)
p < 0.05;
**p < 0.01;
***p < 0.001;
****p < 0.00001;
*****p < 0.000001
Analytes regulated by type I TFN are highlighted in bold font in Table 3.

Down-regulated analytes included IFN-ω, angiotensin converting enzyme (ACE), ACE-2, the chemotactic cytokine (chemokine) CCL20, the growth factor FGF-2, and soluble growth factor receptors (FGF R3 and PDGF-RA). Up-regulated analytes included several cytokine polypeptides (IL-5, IL-6, IL-15, IL-18, BDNF, and GDNF), the cytokine receptors IL-2SRA and TGF-B RIII, matrix metalloproteinase 7 (MMP7), and the adhesion molecule ICAM-3. Twelve of the 23 up-regulated analytes identified were chemokines, including representatives of both the CC- and CXC-families (Zlotnik and Yoshie, Immunity 12:121-127 (2000)).

To validate a subset of the results obtained using the antibody array platform, the levels of two of the chemokines, CCL2 and CXCL9, were measured in 40 serum samples (15 IFN-hi, 12 IFN-lo, and 13 control samples) using Protein Multiplex Immunoassays (Biosource, Camarillo, Calif.) coupled with xMAP technology (Luminex, Austin, Tex.). Samples were analyzed in duplicate, and calibrated recombinant polypeptides were used to generate standard curves. The average coefficient of variance for duplicates was 10.7%. Linear regression was used to calculate correlation coefficients between results obtained using the antibody array platform and the Luminex assay. These analyses indicated that there was a high level of concordance between the two platforms (FIG. 2).

Concentration values for each analyte listed in Table 3 were normalized to the mean of the controls, log₂ transformed, subjected to unsupervised hierarchical clustering (CLUSTER), and visualized using TREEVIEW (Eisen et al., Proc Natl Acad Sci USA 95:14863-14868 (1998)). Hierarchical clustering analysis revealed that many of these polypeptides were coordinately dysregulated in the serum of a large percentage of IFN-hi patients and in the serum of a smaller fraction of IFN-lo patients.

To identify analytes that are regulated by type I IFN, gene expression experiments were performed in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Lymphocyte Separation Medium (Mediatech Cellgro, Herndon, Va.). PBMCs (2 million cells/mL) were resuspended in complete medium (RPMI 1640, 2 mM L-glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin) with 10% autologous plasma. Cells were incubated for 6 or 24 hours with medium alone, or with IFNα/IFNβ (1000 units/mL each; R&D Systems, Minneapolis, Minn.). Total RNA was isolated from the cells and converted to cRNA (Ambion, Austin, Tex.), which was hybridized to Affymetrix U133A gene expression arrays. The gene expression data were analyzed using Affymetrix Microarray Suite 5.0 software. Transcripts were considered to be regulated by IFN if their mean expression values changed by greater than 2-fold in cells stimulated with IFN for 6 or 24 hours compared to corresponding control cells that were not stimulated, and if the difference in expression value was greater than 500 Affymetrix units.

For the analytes listed in Table 3, gene expression data from the in vitro experiments, in which normal PBMCs were stimulated with type I IFN for 6 and 24 hours, were clustered as described above. The data were normalized to control conditions (i.e., incubation with medium-alone). The hierarchical clustering analysis identified analytes that were transcriptionally regulated by type I IFN, as determined by gene expression arrays. Analytes regulated by type I IFN are highlighted in bold font in Table 3. Most of the chemokines identified are inducible by type I IFN. Seven of the 11 analytes that differed in serum levels between IFN-hi and IFN-lo SLE cases were IFN-regulated chemokines.

These data indicate that serum levels of IFN-regulated chemokines are increased and serum polypeptide profiles are broadly dysregulated in many SLE patients. In addition, the highest levels of IFN-inducible analytes are found in patients carrying the IFN blood cell gene signature.

For the majority of individual analytes, there was a poor correlation between the level of gene expression observed in blood and the polypeptide level measured in serum (FIG. 3). In contrast, the IFN-regulated chemokine polypeptide levels were generally highly correlated with the presence of IFN-responsive gene transcripts in blood (UN gene score; Table 4). The IFN gene score was calculated based on expression of 82 IFN-inducible transcripts measured by concurrent whole blood gene expression microarrays, as described herein.

Clinical data, including disease activity indices (SLEDAI, SLAM-R) and laboratory results (anti-DNA antibodies (Abs), complement levels, erythrocyte sedimentation rates (ESR), white blood cell counts, hematocrit), were compared with analyte data using linear regression analysis (Pearson's correlation). One of the IFN-lo cases had insufficient clinical data and was excluded from the regression analyses. To determine the statistical significance of each comparison, random permutation analysis was performed to define the p-value thresholds.

Twenty analytes exhibited one or more significant correlations with clinical measures of disease activity (Table 4). Many of the IFN-regulated analytes exhibited strong positive correlations with the SLEDAI and the SLAM-R, two validated measures of global disease activity, as well as with the erythrocyte sedimentation rate (ESR) and titers of anti-dsDNA antibodies. The IFN-regulated analytes were negatively correlated with hematocrit (HCT) and complement C3 levels and exhibited similar negative trends with complement C4 levels and WBC counts.

An IFN-regulated chemokine polypeptide score was derived from the normalized serum levels of seven IFN-regulated chemokines: CCL2 (MCP-1), CCL3 (MIP-1α), CCL8 (MCP-2), CCL19 (MIP-3β), CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC). The concentration values were normalized across all samples so that the maximum value for any analyte was 1.0, and the values for each sample were summed to derive the final score (FIG. 4). The chemokine polypeptide score reflected the trends observed with the individual IFN-inducible analytes and exhibited stronger associations than any single analyte (Table 4). The chemokine polypeptide score was also more highly correlated with disease activity, as measured by SLEDAI, SLAM-R, ESR, and anti-DNA antibodies, than the IFN gene score (Table 4). These results indicate that there are striking clinical correlations between analytes, especially those that are IFN-regulated, and various measures of SLE disease activity.

TABLE 4
Association of IFN-regulated chemokines with SLE disease activity
	IFN Gene				Anti-DNA
Analyte	Score	SLEDAI	SLAM-R	ESR	Abs	HCT	C3	C4	WBC
ACE-2	−0.41*	−0.24	−0.20	−0.08	−0.25	−0.28	0.28	0.23	0.07
FGF R3	−0.13	−0.10	0.00	0.07	−0.24	0.06	0.44*	0.23	0.30
MMP7	0.00	0.46*	0.21	0.22	0.32	−0.43**	−0.16	0.14	−0.17
TGF-B RIII	0.04	0.38*	0.17	0.17	0.45*	−0.05	−0.06	0.05	0.05
CCL17 (TARC)	0.05	0.19	0.14	−0.01	0.03	0.03	0.12	0.24	0.38*
GDNF	0.12	0.45*	0.44**	0.44**	0.39*	−0.31*	−0.13	−0.19	0.01
CXCL2 (GROB)	0.12	0.39*	0.30	0.15	0.22	−0.15	0.15	0.29	0.17
IL-5	0.17	0.40*	0.40*	0.24	0.35	−0.31*	−0.09	−0.07	0.08
IL-6	0.18	0.26	0.37*	0.40*	0.27	−0.32*	0.01	−0.13	0.11
IL-15	0.20	0.63***	0.48**	0.21	0.53**	−0.44**	−0.26	−0.21	−0.15
IL-18	0.42*	0.10	0.40*	0.50**	0.11	−0.13	−0.05	0.14	0.06
BDNF	0.45**	0.12	0.05	0.00	0.12	0.14	−0.29	−0.15	−0.03
CCL8 (MCP-2)	0.46**	0.52**	0.60***	0.62***	0.68***	−0.52**	−0.43*	−0.32	−0.30
ICAM-3	0.48**	0.38*	0.55**	0.54**	0.20	−0.62***	−0.41*	−0.31	−0.50**
CCL2 (MCP-1)	0.48**	0.35*	0.39*	0.43*	0.57**	−0.21	−0.40*	−0.33*	−0.19
CXCL9 (MIG)	0.54**	0.17	0.48**	0.56***	−0.02	−0.34*	−0.17	−0.08	−0.24
CXCL11 (I-TAC)	0.56***	0.42*	0.67****	0.70****	0.60**	−0.56***	−0.42*	−0.29	−0.32
CXCL10 (IP-10)	0.58***	0.37*	0.50**	0.56***	0.48*	−0.23	−0.40*	−0.33*	−0.31
CCL19 (MIP-3B)	0.59***	0.63***	0.57***	0.33*	0.38*	−0.39*	−0.41*	−0.29	−0.37*
CCL3 (MIP-1A)	0.61***	0.59**	0.68****	0.60***	0.61**	−0.56***	−0.52**	−0.45*	−0.32
Chemokine	0.73****	0.57**	0.72****	0.72****	0.61**	−0.46**	−0.49**	−0.37*	−0.32
Protein Score
IFN Gene Score		0.43*	0.68****	0.52**	0.35	−0.26	−0.51**	−0.39*	−0.40*
*p < 0.05;
**p < 0.01;
***p < 0.001;
****p < 0.00001

Studies were then conducted to determine whether the chemokine levels measured in serum were correlated with specific organ system involvement. The patients were divided based on the presence or absence of organ system disease activity at the time of the visit (renal, serositis, hematologic, and skin), and levels of individual chemokines were compared (Tables 5 and 6). CXCL11 (I-TAC), CXCL13 (BLC), CXCL10 (IP-10) and CCL3 (MIP-1A) were present at significantly higher levels in serum of patients with active renal disease than those without. Levels of CCL8 (MCP-2), CCL2 (MCP-1) and CXCL2 (GROB) trended towards significance. The overall chemokine score was also elevated in patients with renal disease as compared to those without (Table 5).

Higher levels of CCL17 (TARC), CXCL10 (IP-10) and CCL2 (MCP-1) were found in the small subset of patients (N=4) with active serositis. A negative correlation was observed between levels of CCL20 (MIP-3A), CCL17 (TARC), CXCL2 (GROB) and CXCL8 (IL-8) and active hematologic system involvement (mostly thrombocytopenia; Table 5), and several additional chemokines (CXCL13, CCL3 and CXCL11) trended towards significance. No significant correlations were observed for patients with skin disease (Table 6). In a reciprocal approach, the 30 patients were ranked by serum levels of individual chemokines, and each group was divided evenly into chemokine ‘X’ high and low groups for comparison of clinical features. These data generally mirrored the findings shown in Table 5, demonstrating that many chemokines were associated with nephritis in this sample, and that lower chemokine levels were generally observed in individuals with hematologic involvement.

TABLE 5
Chemokine levels and clinical features of SLE
			Mean fold
			change
	Mean ± SD	Mean ± SD	(pos/neg)	P
Renal
	Pos	Neg
	(N = 10)	(N = 20)
CXCL11 (I-TAC)	288 ± 196	162 ± 67	1.77	0.014
Chemokine score	2.7 ± 1.5	1.7 ± 0.7	1.56	0.022
CXCL13 (BLC)	289 ± 191	169 ± 101	1.71	0.032
CXCL10 (IP-10)	127 ± 127	52 ± 59	2.43	0.045
CCL3 (MIP-1A)	429 ± 147	340 ± 87	1.26	0.047
CCL8 (MCP-2)	52 ± 37	32 ± 19	1.63	0.057
CCL2 (MCP-1)	136 ± 148	71 ± 56	1.92	0.090
CXCL2 (GROB)	1322 ± 933	742 ± 824	1.78	0.093
Serositis
	Pos	Neg
	(N = 4)	(N = 26)
CCL17 (TARC)	128 ± 107	59 ± 40	2.16	0.019
CXCL10 (IP-10)	172 ± 179	61 ± 61	2.84	0.023
CCL2 (MCP-1)	175 ± 181	80 ± 79	2.21	0.070
Hematologic
	Pos	Neg
	(N = 7)	(N = 23)
CCL20 (MIP-3A)	67 ± 9	83 ± 15	0.81	0.013
CCL17 (TARC)	26 ± 13	81 ± 58	0.33	0.020
CXCL2 (GROB)	332 ± 220	1119 ± 937	0.30	0.038
CXCL8 (IL-8)	5 ± 2	10 ± 6	0.50	0.038
CXCL13 (BLC)	120 ± 19	237 ± 157	0.51	0.062
CCL3 (MIP-1A)	302 ± 72	390 ± 120	0.77	0.076
CXCL11 (I-TAC)	128 ± 28	227 ± 147	0.56	0.089
Chemokine score	1.4 ± 0.4	2.3 ± 1.2	0.64	0.095

TABLE 6
Serum chemokine levels and specific SLE organ system involvement
	AVG	SD	AVG	SD	P
	Renal pos		Renal neg
	(N = 10)a		(N = 20)
	CXCL13 (BLC)b	289	191	169	101	0.03
	CXCL2 (GROB)	1322	933	742	824	0.1
	CXCL8 (IL-8)	10	7	7	4	0.2
	CXCL10 (IP-10)	127	127	52	59	0.05
	CXCL11 (I-TAC)	288	196	162	67	0.01
	CCL2 (MCP-1)	136	148	71	56	0.1
	CCL8 (MCP-2)	52	37	32	19	0.1
	CCL7 (MCP-3)	53	19	45	11	0.2
	CXCL9 (MIG)	168	189	106	104	0.3
	CCL3 (MIP-1A)	429	147	340	87	0.05
	CCL20 (MIP-3A)	84	13	76	16	0.2
	CCL19 (MIP-3B)	190	107	142	93	0.2
	CCL17 (TARC)	71	43	67	62	0.8
	CK score (0-7)	2.7	1.5	1.7	0.7	0.02
	Sero		Sero neg
	pos (N = 4)		(N = 26)
	CXCL13 (BLC)	301	203	195	135	0.2
	CXCL2 (GROB)	961	396	932	948	1.0
	CXCL8 (IL-8)	12	10	8	4	0.1
	CXCL10 (IP-10)	172	179	61	61	0.02
	CXCL11 (I-TAC)	278	200	193	125	0.3
	CCL2 (MCP-1)	175	181	80	79	0.1
	CCL8 (MCP-2)	55	55	36	21	0.2
	CCL7 (MCP-3)	43	9	49	15	0.5
	CXCL9 (MIG)	127	75	127	147	1.0
	CCL3 (MIP-1A)	445	55	358	119	0.2
	CCL20 (MIP-3A)	79	19	79	15	1.0
	CCL19 (MIP-3B)	143	92	161	101	0.7
	CCL17 (TARC)	128	107	59	40	0.02
	CK score (0-7)	2.8	1.8	1.9	1.0	0.2
	Heme pos		Heme neg
	(N = 7)		(N = 23)
	CXCL13 (BLC)	120	19	237	157	0.1
	CXCL2 (GROB)	332	220	1119	937	0.04
	CXCL8 (IL-8)	5	2	10	6	0.04
	CXCL10 (IP-10)	39	18	88	102	0.3
	CXCL11 (I-TAC)	128	28	227	147	0.1
	CCL2 (MCP-1)	39	25	109	107	0.1
	CCL8 (MCP-2)	24	12	43	29	0.1
	CCL7 (MCP-3)	41	12	50	14	0.1
	CXCL9 (MIG)	81	84	141	150	0.3
	CCL3 (MIP-1A)	302	72	390	120	0.1
	CCL20 (MIP-3A)	67	9	83	15	0.01
	CCL19 (MIP-3B)	145	83	162	104	0.7
	CCL17 (TARC)	26	13	81	58	0.02
	CK score (0-7)	1.4	0.4	2.3	1.2	0.1
	Skin pos		Skin neg
	(N = 6)		(N = 24)
	CXCL13 (BLC)	162	43	221	160	0.4
	CXCL2 (GROB)	1335	1281	836	768	0.2
	CXCL8 (IL-8)	6	3	9	6	0.2
	CXCL10 (IP-10)	90	84	73	96	0.7
	CXCL11 (I-TAC)	178	63	211	149	0.6
	CCL2 (MCP-1)	131	113	83	95	0.3
	CCLX (MCP-2)	41	23	38	29	0.8
	CCL7 (MCP-3)	43	10	49	15	0.4
	CXCL9 (MIG)	105	75	133	151	0.7
	CCL3 (MIP-1A)	360	102	372	121	0.8
	CCL20 (MIP-3A)	86	15	77	15	0.2
	CCL19 (MIP-3B)	187	145	151	86	0.4
	CCL17 (TARC)	69	34	68	61	1.0
	CK score (0-7)	2.2	0.9	2.0	1.2	0.8
	aFor each chemokine and the chemokine score (CK score), an unpaired T-Test was used to determine differences in chemokine levels between patients positive (pos) or negative (neg) for renal, serositis, hematologic, and skin manifestations.
	bSerum chemokine levels reported in pg/ml.

The dataset was expanded to validate candidate SLE biomarkers, including interferon-regulated chemokines, in patient serum. Luminex bead-based technology was used to assay candidate SLE biomarkers in a group of 80 SLE patients with longitudinal visits (˜400 total samples). Serum was obtained and immediately treated with protease inhibitors. Longitudinal samples were available from most patients (average number of visits per patient ˜5), and all patients were evaluated by the same examining physician at every visit. Luminex immunoassays were used to quantitate 8 serum proteins identified as potential SLE biomarkers, as described above. As an initial analysis, a single visit from each patient was used to rank patients from highest to lowest concentration for each protein. Patients in the top and bottom quartiles (n=20 for each) were then compared for disease activity (SLEDAI, PGA, SLAM, BILAG) and other clinical features (DNA abs, ESR, complement, HCT, and WBC count) using unpaired Student's t-test.

Seven proteins showed significant (p<0.05) differences in SLE disease activity as measured by SLAM or SLEDAI, with the interferon-regulated chemokines MCP-2 (SLAM p<0.0006) and IP-10 (SLAM p<0.006, SLEDAI p<0.0002) exhibiting the most significant differences (Tables 7-10). Several laboratory measures including ESR, WBC count, low C3 and C4, and DNA abs were also significantly different between the groups (Table 7). Of particular interest were chemokines IP-10 (ESR p<0.000002, WBC count p<0.003), ENA-78 (ESR p<0.0001) and I-TAC (low C3 p<0.03, low C4 p<0.02, DNA abs p<0.02). Similar results were obtained when comparing protein levels between patient groups based on their ranking by clinical variables (Tables 8-10).

TABLE 7
Clinical feature p-values of the top and bottom 25% of patients as
ranked by analyte levels
	Disease Activity
	PGA	C: joints	B: renal	SLEDAI	BILAG	SLAM-R
TNF-RI	0.38	0.92	0.01*	0.25	0.06	0.01
MCP-2	0.06	0.16	0.02	0.08	0.54	0.0006
IP-10	0.53	0.63	0.76	0.0002	0.24	0.006
MIP-1B	0.98	0.31	0.64	0.06	0.91	0.02
MMP-7	0.36	0.60	0.06	0.06	0.03	0.03
MCP-1	0.38	0.11	0.44	0.15	0.56	0.61
ENA-78	0.14	0.01	0.006	0.08	0.67	0.02
I-TAC	0.52	0.89	0.09	0.15	0.34	0.01
	Lab Tests
	HCT	WBC	LYMPH	ESR	C3	C4	DNAP
TNF-RI	0.86	0.30	0.88	0.01*	0.92	0.67	0.66
MCP-2	0.03	0.02	0.08	0.00002	0.22	0.18	0.41
IP-10	0.001	0.003	0.02	0.000002	0.03	0.01	0.07
MIP-1B	0.16	0.06	0.29	0.03	0.60	0.64	0.03
MMP-7	0.20	0.49	0.86	0.14	0.47	0.14	0.76
MCP-1	0.32	0.93	0.01	0.93	0.02	0.12	0.02
ENA-78	0.01	0.53	0.02	0.0001	0.01	0.04	0.28
I-TAC	0.0004	0.08	0.02	0.003	0.03	0.02	0.03
*Underlined values indicate p < 0.05

TABLE 8
Analyte level p-values of the top and bottom 25% of patients as
ranked by analyte levels
	SLEDAI	SLAM	ESR
	TNF-RI	0.10	0.01*	0.01
	MCP-2	0.07	0.02	0.0007
	IP-10	0.01	0.08	0.01
	MIP-1B	0.71	0.74	0.30
	MMP-7	0.20	0.07	0.33
	MCP-1	0.09	0.11	0.34
	ENA-78	0.09	0.10	0.0015
	I-TAC	0.10	0.02	0.0068
	*Underlined values indicate p < 0.05

TABLE 9
Analyte level p-values in active vs. inactive patients
		Definition 1	Definition 2
	TNF-RI	0.10	0.0061*
	MCP-2	0.03	0.01
	IP-10	0.02	0.38
	MIP-1B	0.16	0.20
	MMP-7	0.05	0.0091
	MCP-1	0.04	0.17
	ENA-78	0.23	0.25
	I-TAC	0.07	0.03
	*Underlined values indicate p < 0.05

TABLE 10
Average analyte level
		SLEDAI	SLEDAI	Slam	Slam
		active	inactive	active	inactive
	TNF-RI	2846	1913	2897*	1666
	MCP-2	62	35	62	35
	IP-10	260	53	202	126
	MIP-1B	212	94	160	103
	MMP-7	12923	7529	12696	6254
	MCP-1	389	178	313	215
	ENA-78	4777	2703	3725	2188
	I-TAC	505	223	573	204
	*Underlined values indicate p < 0.05

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for identifying a mammal having systemic lupus erythematosus, said method comprising (a) determining whether or not serum from a mammal comprises a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4, and (b) classifying said mammal as having systemic lupus erythematosus if said serum comprises said signature and classifying said mammal as not having systemic lupus erythematosus if said serum does not comprise said signature.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1, wherein said method comprises determining whether or not said serum comprises said activity signature 1.

4. The method of claim 1, wherein said method comprises determining whether or not said serum comprises said activity signature 2.

5. The method of claim 1, wherein said method comprises determining whether or not said serum comprises said activity signature 3.

6. The method of claim 1, wherein said method comprises determining whether or not said serum comprises said activity signature 4.

7. A method for assessing systemic lupus erythematosus disease activity, said method comprising (a) determining whether or not serum from a mammal comprises a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4, and (b) classifying said mammal as having active systemic lupus erythematosus disease if said serum comprises said signature and classifying said mammal as not having active systemic lupus erythematosus disease if said serum does not comprise said signature.

8. The method of claim 7, wherein said mammal is a human.

9. The method of claim 7, wherein said method comprises determining whether or not said serum comprises said activity signature 1.

10. The method of claim 7, wherein said method comprises determining whether or not said serum comprises said activity signature 2.

11. The method of claim 7, wherein said method comprises determining whether or not said serum comprises said activity signature 3.

12. The method of claim 7, wherein said method comprises determining whether or not said serum comprises said activity signature 4.

13. A method for determining whether or not a mammal is likely to experience active systemic lupus erythematosus disease, said method comprising (a) determining whether or not serum from a mammal comprises a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4, and (b) classifying said mammal as being likely to experience said active systemic lupus erythematosus disease if said serum comprises said signature and classifying said mammal as not being likely to experience said active systemic lupus erythematosus disease if said serum does not comprise said signature.

14. The method of claim 13, wherein said mammal is a human.

15. The method of claim 13, wherein said method comprises determining whether or not said serum comprises said activity signature 1.

16. The method of claim 13, wherein said method comprises determining whether or not said serum comprises said activity signature 2.

17. The method of claim 13, wherein said method comprises determining whether or not said serum comprises said activity signature 3.

18. The method of claim 13, wherein said method comprises determining whether or not said serum comprises said activity signature 4.

19. A method for assessing effectiveness of a treatment for systemic lupus erythematosus disease, said method comprising determining whether or not a mammal having systemic lupus erythematosus disease and having received a treatment for said systemic erythematosus disease comprises serum comprising a signature selected from the group consisting of an activity signature 1, an activity signature 2, an activity signature 3, and an activity signature 4 to a level less than the level present in an earlier obtained serum sample from said mammal, wherein the presence of said serum indicates that said treatment is effective.

20. The method of claim 19, wherein said mammal is a human.

21. The method of claim 19, wherein said method comprises determining whether or not said serum comprises said activity signature 1.

22. The method of claim 19, wherein said method comprises determining whether or not said serum comprises said activity signature 2.

23. The method of claim 19, wherein said method comprises determining whether or not said serum comprises said activity signature 3.

24. The method of claim 19, wherein said method comprises determining whether or not said serum comprises said activity signature 4.