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1.  Discovery of Biomarkers for Systemic Lupus Erythematosus Using a Library of Synthetic Autoantigen Surrogates 
Journal of immunological methods  2013;402(0):23-34.
Antibodies to a wide range of self-antigens, including those directed against nucleic acids or nucleic acid-binding proteins are the essential biomarkers for diseases such as systemic lupus erythematosus (SLE). Highly complex libraries of nonamers consisting of N-substituted glycines (peptoids) were screened for compounds that bound IgG from patients with SLE and earlier, incomplete autoimmune syndromes. Peptoids were identified that could identify subjects with SLE and related syndromes with a high sensitivity (70%) and specificity (97.5%). Immobilized peptoids were used to isolate IgG from both healthy subjects and SLE patients that reacted with known RNA-binding proteins. In the case of SLE patients, the peptoid-purified IgG reacted with several autoantigens, suggesting that the peptoids are capable of interacting with multiple, structurally similar molecules. These results show that the measurement of IgG binding to peptoids can identify subjects with high levels of pathogenic autoantibodies.
doi:10.1016/j.jim.2013.11.004
PMCID: PMC3946867  PMID: 24269750
3.  SLE Peripheral Blood B Cell, T Cell and Myeloid Cell Transcriptomes Display Unique Profiles and Each Subset Contributes to the Interferon Signature 
PLoS ONE  2013;8(6):e67003.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by defective immune tolerance combined with immune cell hyperactivity resulting in the production of pathogenic autoantibodies. Previous gene expression studies employing whole blood or peripheral blood mononuclear cells (PBMC) have demonstrated that a majority of patients with active disease have increased expression of type I interferon (IFN) inducible transcripts known as the IFN signature. The goal of the current study was to assess the gene expression profiles of isolated leukocyte subsets obtained from SLE patients. Subsets including CD19+ B lymphocytes, CD3+CD4+ T lymphocytes and CD33+ myeloid cells were simultaneously sorted from PBMC. The SLE transcriptomes were assessed for differentially expressed genes as compared to healthy controls. SLE CD33+ myeloid cells exhibited the greatest number of differentially expressed genes at 208 transcripts, SLE B cells expressed 174 transcripts and SLE CD3+CD4+ T cells expressed 92 transcripts. Only 4.4% (21) of the 474 total transcripts, many associated with the IFN signature, were shared by all three subsets. Transcriptional profiles translated into increased protein expression for CD38, CD63, CD107a and CD169. Moreover, these studies demonstrated that both SLE lymphoid and myeloid subsets expressed elevated transcripts for cytosolic RNA and DNA sensors and downstream effectors mediating IFN and cytokine production. Prolonged upregulation of nucleic acid sensing pathways could modulate immune effector functions and initiate or contribute to the systemic inflammation observed in SLE.
doi:10.1371/journal.pone.0067003
PMCID: PMC3691135  PMID: 23826184
4.  IgG and IgM autoantibody differences in discoid and systemic lupus patients 
Systemic lupus (SLE) patients with discoid lupus (DLE) were reported to have milder disease. To test this observation, we employed sandwich arrays containing 98 autoantigens to compare autoantibody profiles of SLE subjects without DLE (DLE−SLE+) (N=9), SLE subjects with DLE (DLE+SLE+) (N=10), DLE subjects without SLE (DLE+SLE−) (N=11), and healthy controls (N=11). We validated differentially expressed autoantibodies using immunoassays in DLE−SLE+ (N=18), DLE+SLE+ (N=17), DLE+SLE− (N=23), and healthy subjects (N=22). Arrays showed 15 IgG autoantibodies (ten against nuclear antigens) and four IgM autoantibodies that were differentially expressed (q-value<0.05). DLE−SLE+ subjects had higher IgG autoantibodies against dsDNA, ssDNA, dsRNA, histone H2A and H2B, and SS-A (52 kDa) than all other groups including DLE+SLE+ subjects (p<0.05). Immunoassays measuring anti-dsDNA, -ssDNA, and -SS-A (52 kDa) IgG autoantibodies showed similar trends (p<0.05). Healthy and DLE+SLE−subjects expressed higher IgM autoantibodies against alpha beta crystallin, lipopolysaccharide, heat shock cognate 70, and desmoglein-3 than DLE+SLE+ and DLE−SLE+ subjects. IgG:IgM ratios of autoantibodies against nuclear antigens progressively rose from healthy to DLE−SLE+ subjects. In conclusion, lower IgG autoantibodies against nuclear antigens in DLE+SLE+ versus DLE−SLE+ subjects suggest that DLE indicates lower disease severity. Higher IgM autoantibodies against selected antigens in healthy and DLE+SLE−subjects may be non-pathogenic.
doi:10.1038/jid.2012.207
PMCID: PMC3465644  PMID: 22763789
5.  Autoantibody profiling to follow evolution of lupus syndromes 
Arthritis Research & Therapy  2012;14(4):R174.
Introduction
Identification of patients who are in early stages of lupus is currently done through clinical evaluation and is not greatly facilitated by available diagnostic tests. Profiling for patient characteristics and antibody specificities that predict disease would enhance the ability of physicians to identify and treat early cases prior to onset of organ damaging illness.
Methods
A group of 22 patients with 4 or fewer diagnostic criteria for lupus were studied for changes in clinical and autoantibody profiles after a mean follow up period of 2.4 years. An array with more than 80 autoantigens was used to profile immunoglobulin G (IgG) and immunoglobulin M (IgM) autoantibodies. Correlations with clinical disease progression were examined.
Results
3 of the 22 patients (14%) added sufficient criteria during follow up to satisfy a diagnosis of systemic lupus erythematosus (SLE) or to acquire a diagnosis of SLE renal disease. Patients who progressed were all females and were younger than those who did not progress (P=0.00054). IgG but not IgM autoreactivity showed greater increases in the progressor group than in the non-progressor group (P=0.047). IgG specificities that were higher at baseline in progressors included proliferating cell nuclear antigen (PCNA), beta 2 microglobulin, C1q and hemocyanin (P<0.019). Progressors had significant increases in La/SSB and liver cytosol type 1 (LC1) IgG autoantibodies over the period of evaluation (P≤0.0072). A quantitative risk profile generated from baseline demographic and autoantibody variables yielded highly different scores for the progressor and non-progressor groups (P=1.38 × 10-7)
Conclusions
In addition to demographic features, autoantibody profiles using an expanded array of specificities were correlated with the risk of progressive disease in patients with lupus. These findings suggest the feasibility of developing a simple diagnostic that could be applied by nonspecialists to screen for lupus and permit effective triage for specialty care.
doi:10.1186/ar3927
PMCID: PMC3580568  PMID: 22838636
6.  The Lupus Family Registry and Repository 
Rheumatology (Oxford, England)  2010;50(1):47-59.
The Lupus Family Registry and Repository (LFRR) was established with the goal of assembling and distributing materials and data from families with one or more living members diagnosed with SLE, in order to address SLE genetics. In the present article, we describe the problems and solutions of the registry design and biometric data gathering; the protocols implemented to guarantee data quality and protection of participant privacy and consent; and the establishment of a local and international network of collaborators. At the same time, we illustrate how the LFRR has enabled progress in lupus genetics research, answering old scientific questions while laying out new challenges in the elucidation of the biologic mechanisms that underlie disease pathogenesis. Trained staff ascertain SLE cases, unaffected family members and population-based controls, proceeding in compliance with the relevant laws and standards; participant consent and privacy are central to the LFRR’s effort. Data, DNA, serum, plasma, peripheral blood and transformed B-cell lines are collected and stored, and subject to strict quality control and safety measures. Coded data and materials derived from the registry are available for approved scientific users. The LFRR has contributed to the discovery of most of the 37 genetic associations now known to contribute to lupus through 104 publications. The LFRR contains 2618 lupus cases from 1954 pedigrees that are being studied by 76 approved users and their collaborators. The registry includes difficult to obtain populations, such as multiplex pedigrees, minority patients and affected males, and constitutes the largest collection of lupus pedigrees in the world. The LFRR is a useful resource for the discovery and characterization of genetic associations in SLE.
doi:10.1093/rheumatology/keq302
PMCID: PMC3307518  PMID: 20864496
Systemic lupus erythematosus; Registry; Repository; Autoimmune diseases; Genetics; Heritability; Genome-wide association studies; Linkage analysis; Minorities; Women
7.  Risk factors for ANA positivity in healthy persons 
Introduction
The finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.
Methods
Biological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles.
Results
Overall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature.
Conclusions
Risks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.
doi:10.1186/ar3271
PMCID: PMC3132017  PMID: 21366908
8.  Novel sequence feature variant type analysis of the HLA genetic association in systemic sclerosis 
Human Molecular Genetics  2009;19(4):707-719.
We describe a novel approach to genetic association analyses with proteins sub-divided into biologically relevant smaller sequence features (SFs), and their variant types (VTs). SFVT analyses are particularly informative for study of highly polymorphic proteins such as the human leukocyte antigen (HLA), given the nature of its genetic variation: the high level of polymorphism, the pattern of amino acid variability, and that most HLA variation occurs at functionally important sites, as well as its known role in organ transplant rejection, autoimmune disease development and response to infection. Further, combinations of variable amino acid sites shared by several HLA alleles (shared epitopes) are most likely better descriptors of the actual causative genetic variants. In a cohort of systemic sclerosis patients/controls, SFVT analysis shows that a combination of SFs implicating specific amino acid residues in peptide binding pockets 4 and 7 of HLA-DRB1 explains much of the molecular determinant of risk.
doi:10.1093/hmg/ddp521
PMCID: PMC2807365  PMID: 19933168
9.  SEQUENCE FEATURE VARIANT TYPE (SFVT) ANALYSIS OF THE HLA GENETIC ASSOCIATION IN JUVENILE IDIOPATHIC ARTHRITIS 
The immune response HLA class II DRB1 gene provides the major genetic contribution to Juvenile Idiopathic Arthritis (JIA), with a hierarchy of predisposing through intermediate to protective effects. With JIA, and the many other HLA associated diseases, it is difficult to identify the combinations of biologically relevant amino acid (AA) residues directly involved in disease due to the high level of HLA polymorphism, the pattern of AA variability, including varying degrees of linkage disequilibrium (LD), and the fact that most HLA variation occurs at functionally important sites. In a subset of JIA patients with the clinical phenotype oligoarticular-persistent (OP), we have applied a recently developed novel approach to genetic association analyses with genes/proteins sub-divided into biologically relevant smaller sequence features (SFs), and their “alleles” which are called variant types (VTs). With SFVT analysis, association tests are performed on variation at biologically relevant SFs based on structural (e.g., beta-strand 1) and functional (e.g., peptide binding site) features of the protein. We have extended the SFVT analysis pipeline to additionally include pairwise comparisons of DRB1 alleles within serogroup classes, our extension of the Salamon Unique Combinations algorithm, and LD patterns of AA variability to evaluate the SFVT results; all of which contributed additional complementary information. With JIA-OP, we identified a set of single AA SFs, and SFs in which they occur, particularly pockets of the peptide binding site, that account for the major disease risk attributable to HLA DRB1. These are (in numeric order): AAs 13 (pockets 4 and 6), 37 and 57 (both pocket 9), 67 (pocket 7), 74 (pocket 4), and 86 (pocket 1), and to a lesser extent 30 (pockets 6 and 7) and 71 (pockets 4, 5, and 7).
PMCID: PMC2958177  PMID: 19908388
10.  Ethical and Practical Issues Associated with Aggregating Databases 
PLoS Medicine  2008;5(9):e190.
David Karp and colleagues discuss the ethical and practical concerns that arise when data are shared in aggregated databases.
doi:10.1371/journal.pmed.0050190
PMCID: PMC2553818  PMID: 18816162
11.  A New Type of Metal Recognition by Human T Cells 
The Journal of Experimental Medicine  2003;197(10):1345-1353.
In spite of high frequencies of metal allergies, the structural basis for major histocompatibility complex (MHC)-restricted metal recognition is among the unanswered questions in the field of T cell activation. For the human T cell clone SE9, we have identified potential Ni contact sites in the T cell receptor (TCR) and the restricting human histocompatibility leukocyte antigen (HLA)-DR structure. The specificity of this HLA-DR–promiscuous VA22/VB17+ TCR is primarily harbored in its α chain. Ni reactivity is neither dependent on protein processing in antigen-presenting cells nor affected by the nature of HLA-DR–associated peptides. However, SE9 activation by Ni crucially depends on Tyr29 in CDR1α, an N-nucleotide–encoded Tyr94 in CDR3α, and a conserved His81 in the HLA-DR β chain. These data indicate that labile, nonactivating complexes between the SE9 TCR and most HLA-DR/peptide conjugates might supply sterically optimized coordination sites for Ni ions, three of which were identified in this study. In such complexes Ni may effectively bridge the TCR α chain to His81 of most DR molecules. Thus, in analogy to superantigens, Ni may directly link TCR and MHC in a peptide-independent manner. However, unlike superantigens, Ni requires idiotypic, i.e., CDR3α-determined TCR amino acids. This new type of TCR–MHC linkage might explain the high frequency of Ni-reactive T cells in the human population.
doi:10.1084/jem.20030121
PMCID: PMC2193783  PMID: 12756270
hypersensitivity; antigen presentation; hapten; T cell receptor; mutation

Results 1-11 (11)