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2.  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.
PMCID: PMC3946867  PMID: 24269750
3.  Overexpression of Membrane-bound Fas Ligand (CD95L) Exacerbates Autoimmune Disease and Renal Pathology in Pristane-induced Lupus 
Loss of function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-Tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces SLE-like disease. While Fas/FasL interactions have been strongly implicated in the activation induced cell death of both lymphocytes and other antigen presenting cells, FasL can also trigger the production of pro-inflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase (MMP) cleavage site in the ectodomain. MMP cleavage inactivates membrane-bound FasL (mFasL) and releases a soluble form, sFasL, reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the pro-apoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation, to produce the ΔCS mouse line. ΔCS mice express higher levels of mFasL than WT mice and fail to release sFasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared to control TMPD-injected BALB/c mice. We found that FasL-deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. By contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in ANA specificity, and markedly increased proteinuria and kidney pathology, compared to controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL pro-inflammatory activity.
PMCID: PMC4219920  PMID: 23918976
4.  IL-21 promotes the production of anti-DNA IgG but is dispensable for kidney damage in lyn−/− mice 
European journal of immunology  2012;43(2):382-393.
The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of SLE, lack an inhibitor of B- and myeloid cell activation. This results in B-cell hyperresponsiveness, plasma cell (PC) accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn−/− mice. We find that IL-21 mRNA is reduced in the spleens of lyn−/−IL-6−/− and lyn−/−Btklo mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for PC accumulation and IgM autoantibodies in lyn−/− mice, it is required for anti-DNA IgG antibodies and some aspects of T cell activation. Surprisingly, GN still develops in lyn−/−IL-21−/− mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA antigens. These studies identify a specific role for IL-21 in the class switching of anti-DNA B-cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn−/− mice.
PMCID: PMC3768150  PMID: 23169140
autoimmunity; Lyn; IL-21; IL-6; autoantibody; glomerulonephritis
5.  Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation 
Oxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN.
The human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 106 hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis.
MSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage.
Our data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.
PMCID: PMC4055015  PMID: 24480247
6.  SNPs in VKORC1 are Risk Factors for Systemic Lupus Erythematosus in Asians 
Arthritis and rheumatism  2013;65(1):211-215.
The increased risk of thrombosis in systemic lupus erythematosus (SLE) may be partially explained by interrelated genetic pathways for thrombosis and SLE. In a case-control analysis, we investigated whether 33 established and novel single nucleotide polymorphisms (SNP) in 20 genes involved in hemostasis pathways that have been associated with deep venous thrombosis in the general population were risk factors for SLE development among Asians.
Patients in the discovery cohort were enrolled in one of two North American SLE cohorts. Patients in the replication cohort were enrolled in one of four Asian or two North American cohorts. SLE cases met American College of Rheumatology classification criteria. We first genotyped 263 Asian SLE and 357 healthy Asian control individuals for 33 SNPs using Luminex multiplex technology in the discovery phase, and then used Taqman and Immunochip assays to examine 5 SNPs in up to an additional 1496 cases and 993 controls in the Replication phase. SLE patients were compared to healthy controls for association with minor alleles in allelic models. Principal components analysis was used to control for intra-Asian ancestry in an analysis of the replication cohort.
Two genetic variants in the gene VKORC1, rs9934438 and rs9923231, were highly significant in both the discovery and replication cohorts: OR(disc) = 2.45 (p=2×10−9), OR(rep) = 1.53 (p=5×10−6) and OR(disc) = 2.40 (p=6×10−9), OR(rep) = 1.53 (p=5×10−6), respectively. These associations were significant in the replication cohort after adjustment for intra-Asian ancestry: rs9934438 OR(adj) = 1.34 (p=0.0029) and rs9923231 OR(adj) = 1.34 (p=0.0032).
Genetic variants in VKORC1, involved in vitamin K reduction and associated with DVT, are associated with SLE development in Asians. These results suggest intersecting genetic pathways for the development of SLE and thrombosis.
PMCID: PMC3670944  PMID: 23124848
systemic lupus erythematosus; single nucleotide polymorphisms; genetic risk factors
7.  The constant region contributes to the antigenic specificity and renal pathogenicity of murine anti-DNA antibodies 
Journal of autoimmunity  2012;39(4):398-411.
Affinity for DNA and cross-reactivity with renal antigens are associated with enhanced renal pathogenicity of lupus autoantibodies. In addition, certain IgG subclasses are enriched in nephritic kidneys, suggesting that isotype may determine the outcome of antibody binding to renal antigens. To investigate if the isotype of DNA antibodies affects renal pathogenicity by influencing antigen binding, we derived IgM, IgG1, IgG2b and IgG2a forms of the PL9–11 antibody (IgG3 anti-DNA) by in vitro class switching or PCR cloning. The affinity and specificity of PL9–11 antibodies for nuclear and renal antigens were analyzed using ELISA, Western blotting, surface plasmon resonance (SPR), binding to mesangial cells, and glomerular proteome arrays. Renal deposition and pathogenicity were assayed in mice injected with PL9–11 hybridomas. We found that PL9–11 and its isotype-switched variants had differential binding to DNA and chromatin (IgG3 > IgG2a > IgG1 > IgG2b > IgM) by direct and competition ELISA, and SPR. In contrast, in binding to laminin and collagen IV the IgG2a isotype actually had the highest affinity. Differences in affinity of PL9–11 antibodies for renal antigens were mirrored in analysis of specificity for glomeruli, and were associated with significant differences in renal pathogenicity in vivo and survival. Our novel findings indicate that the constant region plays an important role in the nephritogenicity of antibodies to DNA by affecting immunoglobulin affinity and specificity. Increased binding to multiple glomerular and/or nuclear antigens may contribute to the renal pathogenicity of anti-DNA antibodies of the IgG2a and IgG3 isotype. Finally, class switch recombination may be another mechanism by which B cell autoreactivity is generated.
PMCID: PMC3656415  PMID: 22841793
Systemic lupus erythematosus (SLE); Lupus nephritis; Anti-DNA antibodies; Isotype switching
8.  Molecular Basis of 9G4 B Cell Autoreactivity in Human Systemic Lupus Erythematosus 
The Journal of Immunology Author Choice  2013;191(10):4926-4939.
9G4+ IgG Abs expand in systemic lupus erythematosus (SLE) in a disease-specific fashion and react with different lupus Ags including B cell Ags and apoptotic cells. Their shared use of VH4-34 represents a unique system to understand the molecular basis of lupus autoreactivity. In this study, a large panel of recombinant 9G4+ mAbs from single naive and memory cells was generated and tested against B cells, apoptotic cells, and other Ags. Mutagenesis eliminated the framework-1 hydrophobic patch (HP) responsible for the 9G4 idiotype. The expression of the HP in unselected VH4-34 cells was assessed by deep sequencing. We found that 9G4 Abs recognize several Ags following two distinct structural patterns. B cell binding is dependent on the HP, whereas anti-nuclear Abs, apoptotic cells, and dsDNA binding are HP independent and correlate with positively charged H chain third CDR. The majority of mutated VH4-34 memory cells retain the HP, thereby suggesting selection by Ags that require this germline structure. Our findings show that the germline-encoded HP is compulsory for the anti–B cell reactivity largely associated with 9G4 Abs in SLE but is not required for reactivity against apoptotic cells, dsDNA, chromatin, anti-nuclear Abs, or cardiolipin. Given that the lupus memory compartment contains a majority of HP+ VH4-34 cells but decreased B cell reactivity, additional HP-dependent Ags must participate in the selection of this compartment. This study represents the first analysis, to our knowledge, of VH-restricted autoreactive B cells specifically expanded in SLE and provides the foundation to understand the antigenic forces at play in this disease.
PMCID: PMC3816606  PMID: 24108696
9.  Enoyl Coenzyme A Hydratase Domain–Containing 2, a Potential Novel Regulator of Myocardial Ischemia Injury 
We reported previously that Brown Norway (BN) rats are more resistant to myocardial ischemia/reperfusion (I/R) injury than are Dahl S (SS) rats. To identify the unique genes differentially expressed in the hearts of these rats, we used DNA microarray analysis and observed that enoyl coenzyme A hydratase–containing domain 2 (ECHDC2) is highly expressed (≈18‐fold) in the SS hearts compared with the BN hearts.
Methods and Results
RT‐PCR, Western blot, and immunohistochemistry analyses verified that ECHDC2 was highly expressed in SS hearts compared with the BN hearts. ECHDC2 gene locates at chromosome 5 of rat and is expressed in mitochondria of the heart, mainly in cardiomyocytes but not in cardiofibroblasts. Overexpression of ECHDC2 in cells increased susceptibility to I/R injury while knockdown of ECHDC2 enhanced resistance to I/R injury. Furthermore, we observed that left anterior descending coronary artery ligation–induced myocardial infarction was more severe in the SS hearts than in the BN hearts or SSBN5 hearts, which was built on SS rats but had the substitution of chromosome 5 from BN rats. We also demonstrated that ECHDC2 did not alter mitochondrial O2 consumption, metabolic intermediates and ATP production. By gas chromatography–mass spectrometry, we found that ECHDC2 overexpression increased the levels of the cellular branched chain amino acids leucine and valine.
ECHDC2, a mitochondrial protein, may be involved in regulating cell death and myocardial injury. Its deficiency in BN rats contributes to their increased resistance to myocardial I/R compared with SS rats. ECHDC2 increases branched chain amino acid metabolism and appears to be a novel regulator linking cell metabolism with cardiovascular disease.
PMCID: PMC3835224  PMID: 24108764
branched amino acid metabolism; cell death; ECHDC2; ischemia/reperfusion injury; myocardial infarction
10.  Kallikrein Transduced Mesenchymal Stem Cells Protect against Anti-GBM Disease and Lupus Nephritis by Ameliorating Inflammation and Oxidative Stress 
PLoS ONE  2013;8(7):e67790.
Previously we have shown that kallikreins (klks) play a renoprotective role in nephrotoxic serum induced nephritis. In this study, we have used mesenchymal stem cells (MSCs) as vehicles to deliver klks into the injured kidneys and have measured their therapeutic effect on experimental antibody induced nephritis and lupus nephritis. Human KLK-1 (hKLK1) gene was transduced into murine MSCs using a retroviral vector to generate a stable cell line, hKLK1-MSC, expressing high levels of hKLK1. 129/svj mice subjected to anti-GBM induced nephritis were transplanted with 106 hKLK1-MSCs and hKLK1 expression was confirmed in the kidneys. Compared with vector-MSCs injected mice, the hKLK1-MSCs treated mice showed significantly reduced proteinuria, blood urea nitrogen (BUN) and ameliorated renal pathology. Using the same strategy, we treated lupus-prone B6.Sle1.Sle3 bicongenic mice with hKLK1-MSCs and demonstrated that hKLK1-MSCs delivery also attenuated lupus nephritis. Mechanistically, hKLK1-MSCs reduced macrophage and T-lymphocyte infiltration into the kidney by suppressing the expression of inflammation cytokines. Moreover, hKLK1 transduced MSCs were more resistant to oxidative stress-induced apoptosis. These findings advance genetically modified MSCs as potential gene delivery tools for targeting therapeutic agents to the kidneys in order to modulate inflammation and oxidative stress in lupus nephritis.
PMCID: PMC3720854  PMID: 23935844
11.  Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci 
Nature genetics  2008;40(2):204-210.
Systemic lupus erythematosus (SLE) is a common systemic autoimmune disease with complex etiology but strong clustering in families (λS = ~30). We performed a genome-wide association scan using 317,501 SNPs in 720 women of European ancestry with SLE and in 2,337 controls, and we genotyped consistently associated SNPs in two additional independent sample sets totaling 1,846 affected women and 1,825 controls. Aside from the expected strong association between SLE and the HLA region on chromosome 6p21 and the previously confirmed non-HLA locus IRF5 on chromosome 7q32, we found evidence of association with replication (1.1 × 10−7 < Poverall < 1.6 × 10−23; odds ratio 0.82–1.62)in four regions: 16p11.2 (ITGAM), 11p15.5 (KIAA1542), 3p14.3 (PXK) and 1q25.1 (rs10798269). We also found evidence for association (P < 1 × 10−5) at FCGR2A, PTPN22 and STAT4, regions previously associated with SLE and other autoimmune diseases, as well as at ≥9 other loci (P < 2 × 10−7). Our results show that numerous genes, some with known immune-related functions, predispose to SLE.
PMCID: PMC3712260  PMID: 18204446
12.  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.
PMCID: PMC3691135  PMID: 23826184
13.  Complete Genome Analysis of Three Acinetobacter baumannii Clinical Isolates in China for Insight into the Diversification of Drug Resistance Elements 
PLoS ONE  2013;8(6):e66584.
The emergence and rapid spreading of multidrug-resistant Acinetobacter baumannii strains has become a major health threat worldwide. To better understand the genetic recombination related with the acquisition of drug-resistant elements during bacterial infection, we performed complete genome analysis on three newly isolated multidrug-resistant A. baumannii strains from Beijing using next-generation sequencing technology.
Methodologies/Principal Findings
Whole genome comparison revealed that all 3 strains share some common drug resistant elements including carbapenem-resistant blaOXA-23 and tetracycline (tet) resistance islands, but the genome structures are diversified among strains. Various genomic islands intersperse on the genome with transposons and insertions, reflecting the recombination flexibility during the acquisition of the resistant elements. The blood-isolated BJAB07104 and ascites-isolated BJAB0868 exhibit high similarity on their genome structure with most of the global clone II strains, suggesting these two strains belong to the dominant outbreak strains prevalent worldwide. A large resistance island (RI) of about 121-kb, carrying a cluster of resistance-related genes, was inserted into the ATPase gene on BJAB07104 and BJAB0868 genomes. A 78-kb insertion element carrying tra-locus and blaOXA-23 island, can be either inserted into one of the tniB gene in the 121-kb RI on the chromosome, or transformed to conjugative plasmid in the two BJAB strains. The third strains of this study, BJAB0715, which was isolated from spinal fluid, exhibit much more divergence compared with above two strains. It harbors multiple drug-resistance elements including a truncated AbaR-22-like RI on its genome. One of the unique features of this strain is that it carries both blaOXA-23 and blaOXA-58 genes on its genome. Besides, an Acinetobacter lwoffii adeABC efflux element was found inserted into the ATPase position in BJAB0715.
Our comparative analysis on currently completed Acinetobacter baumannii genomes revealed extensive and dynamic genome organizations, which may facilitate the bacteria to acquire drug-resistance elements into their genomes.
PMCID: PMC3691203  PMID: 23826102
14.  Evidence for intranasal anti-nuclear autoantibodies in Chronic Rhinosinusitis with Nasal Polyps. 
Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory condition of the nasal passage and paranasal sinuses characterized by Th2 biased inflammation with elevated levels of BAFF, B-lymphocytes, and immunoglobulins. Since high levels of BAFF are associated with autoimmune diseases, we assessed for evidence of autoimmunity in patients with CRS.
The objective of this study was to investigate for the presence of autoantibodies in sinonasal tissue from patients with CRS.
Standardized nasal tissue specimens were collected from patients with CRS and control subjects and assayed for immunoglobulin production, autoantibody levels, tissue distribution of immunoglobulins and binding potential of antibodies in nasal tissue using a multiplexed autoantibody microarray, ELISA and immunofluoresence.
Elevated levels of several specific autoantibodies were found in nasal polyp tissue in comparison with control tissue and inflamed tissue from non-polypoid CRS (CRSsNP) (p<0.05). In particular, nuclear-targeted autoantibodies such as anti-dsDNA IgG and IgA antibodies were found at elevated levels in nasal polyps (p<0.05) and particularly in nasal polyps from patients requiring revision surgery for recurrence. Direct immunofluorescence staining demonstrated diffuse epithelial and sub-epithelial deposition of IgG and increased numbers of IgA secreting plasma cells not seen in control nasal tissue.
Autoantibodies, particularly those against nuclear antigens, are present at locally elevated levels in nasal polyps. The presence of autoantibodies suggests that the microenvironment of a nasal polyp promotes the expansion of self-reactive B-cell clones. While the pathogenicity of these antibodies remains to be elucidated, the presence of elevated anti-dsDNA antibodies is associated with a clinically more aggressive form of CRSwNP requiring repeated surgery.
PMCID: PMC3384688  PMID: 21996343
Chronic Rhinosinusitis; Sinusitis; Nasal Polyps; Autoimmunity; Autoantibodies; Biomarker
15.  Monophosphorylation of CD79a and b ITAM motifs initiates a SHIP-1 phosphatase-mediated inhibitory signaling cascade required for B cell anergy 
Immunity  2011;35(5):746-756.
Anergic B cells are characterized by impaired signaling and activation following aggregation of their antigen receptors (BCR). The molecular basis of this impairment is not understood. In studies reported here Src homology-2 (SH2)-containing inositol 5-phosphatase SHIP-1 and its adaptor Dok-1 were found to be constitutively phosphorylated in anergic B cells, and activation of this inhibitory circuit was dependent on Src-family kinase activity and consequent to biased BCR immunoreceptor tyrosine-based activation motif (ITAM) monophosphorylation. B cell-targeted deletion of SHIP-1 caused severe lupus-like disease. Moreover, absence of SHIP-1 in B cells led to loss of anergy as indicated by restoration of BCR signaling, loss of anergic surface phenotype and production of autoantibodies. Thus chronic BCR signals maintain anergy in part via ITAM monophosphorylation-directed activation of an inhibitory signaling circuit involving SHIP-1 and Dok-1.
PMCID: PMC3232011  PMID: 22078222
16.  Elevated IgG autoantibody production in oligoarticular juvenile idiopathic arthritis may predict a refractory course 
Although oligoarticular juvenile idiopathic arthritis (oJIA) is considered to carry the best prognosis among the JIA subtypes, many children evolve to a chronic course. A few studies have identified clinical risk factors for disease extension, and recent studies have evaluated synovial fluid markers. However, the only biological marker from the serum studied to date is the anti-nuclear antibody (ANA), regarding which there is mixed data regarding prognosis. No studies have evaluated whether additional autoantibodies may affect the articular prognosis of oJIA.
Microarrays containing candidate autoantigens were printed on slides, which were used to profile 36 children with oJIA and 18 controls. Unsupervised clustering analysis was used to identify distinct subgroups of JIA patients. Response to therapy after a mean interval of 4.9 months was evaluated.
Cluster analysis revealed two subgroups of oJIA patients, with identical clustering observed when children with onset over age six were excluded. Cluster 1 had higher levels of multiple autoantibodies compared to both cluster 2 as well as controls, including antibodies against several extracellular matrix (ECM) and nuclear antigens. Although the two patient clusters were similar with respect to clinical features and treatment decisions, children in cluster 1 were less likely to have attained remission by the follow-up visit.
Antibodies against ECM and possibly other antigens may identify a sub-group of children with oJIA who will require more aggressive therapy to attain control of the arthritis.
PMCID: PMC3171611  PMID: 21813070
oligoarticular; juvenile idiopathic arthritis; antibodies
17.  Autoantibody profiling to follow evolution of lupus syndromes 
Arthritis Research & Therapy  2012;14(4):R174.
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.
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.
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)
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.
PMCID: PMC3580568  PMID: 22838636
18.  Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes 
Human Molecular Genetics  2011;20(7):1456-1466.
Adolescent idiopathic scoliosis (AIS) is an unexplained and common spinal deformity seen in otherwise healthy children. Its pathophysiology is poorly understood despite intensive investigation. Although genetic underpinnings are clear, replicated susceptibility loci that could provide insight into etiology have not been forthcoming. To address these issues, we performed genome-wide association studies (GWAS) of ∼327 000 single nucleotide polymorphisms (SNPs) in 419 AIS families. We found strongest evidence of association with chromosome 3p26.3 SNPs in the proximity of the CHL1 gene (P < 8 × 10−8 for rs1400180). We genotyped additional chromosome 3p26.3 SNPs and tested replication in two follow-up case–control cohorts, obtaining strongest results when all three cohorts were combined (rs10510181 odds ratio = 1.49, 95% confidence interval = 1.29–1.73, P = 2.58 × 10−8), but these were not confirmed in a separate GWAS. CHL1 is of interest, as it encodes an axon guidance protein related to Robo3. Mutations in the Robo3 protein cause horizontal gaze palsy with progressive scoliosis (HGPPS), a rare disease marked by severe scoliosis. Other top associations in our GWAS were with SNPs in the DSCAM gene encoding an axon guidance protein in the same structural class with Chl1 and Robo3. We additionally found AIS associations with loci in CNTNAP2, supporting a previous study linking this gene with AIS. Cntnap2 is also of functional interest, as it interacts directly with L1 and Robo class proteins and participates in axon pathfinding. Our results suggest the relevance of axon guidance pathways in AIS susceptibility, although these findings require further study, particularly given the apparent genetic heterogeneity in this disease.
PMCID: PMC3049353  PMID: 21216876
19.  Separate Checkpoints Regulate Splenic Plasma Cell Accumulation and IgG Autoantibody Production in Lyn-deficient Mice 
European journal of immunology  2010;40(7):1897-1905.
Accumulation of plasma cells and autoantibodies against nuclear antigens characterize both human and murine lupus. Understanding how these processes are controlled may reveal novel therapeutic targets for this disease. Mice deficient in Lyn, a negative regulator of B and myeloid cell activity, develop lupus-like autoimmune disease. Here, we show that lyn−/− mice exhibit increased splenic plasmablasts and plasma cells and produce IgM against a wide range of self-antigens. Both events require Btk, a target of Lyn-dependent inhibitory pathways. A Btk-dependent increase in the expression of the plasma cell survival factor IL-6 by lyn−/− splenic myeloid cells was also observed. Surprisingly, IL-6 was not required for plasma cell accumulation or polyclonal IgM autoreactivity in lyn−/− mice. IL-6 was, however, necessary for the production of IgG autoantibodies, which we show are focused in lyn−/− mice towards a limited set of nucleic acid-containing and glomerular autoantigens. A similar uncoupling of plasma cell accumulation from IgG autoantibodies was seen in lyn+/− mice. Plasma cell accumulation and polyclonal IgM autoreactivity are therefore controlled separately from, and are insufficient for, the production of IgG against lupus-associated autoantigens. Regulators of either of these two checkpoints may be attractive therapeutic targets for lupus.
PMCID: PMC3057185  PMID: 20394076
autoimmunity; Lyn; Btk; IL-6; plasma cell
20.  Risk factors for ANA positivity in healthy persons 
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.
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.
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.
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.
PMCID: PMC3132017  PMID: 21366908
21.  Autoantibody Profiles in Two Patients with Non-Autoimmune Muscle Disease Implicate a Role for Gliadin Autoreactivity 
Neuromuscular disorders : NMD  2010;20(3):188-191.
The objective of this case study was to characterize autoreactivity in two patients with non-autoimmune forms of muscle disease who had positivity for antinuclear antibodies (ANA) and Ro (SSA) autoantibodies. Serum samples from these two patients were applied to an autoantigen protein array with more than 70 specificities and were compared to samples from healthy controls and patients with systemic lupus erythematosus. Both myopathy patients had high levels of gliadin autoreactivity in serum and one patient had an overall autoantibody profile with lupus-like features. The findings suggest that some disorders of muscle that are considered nonautoimmune, may in fact have autoimmune features. Further examination of the role of subclinical gluten autoreactivity in the pathogenesis of myopathy syndromes has the potential to suggest improved approaches to diagnosis and treatment of these conditions.
PMCID: PMC2841214  PMID: 20110170
autoantibodies; C1q; dysferlinopathy; gliadin; myopathy
22.  Yaa-autoimmune phenotypes are conferred by an overexpression of TLR7 
European journal of immunology  2008;38(7):1971-1978.
The y-linked autoimmune accelerating (Yaa) locus drives the transition to fatal lupus nephritis when combined with B6.Sle1 in our B6-congenic model of systemic autoimmunity. We and others recently demonstrated that the translocation of a cluster of X-linked genes onto the Y chromosome is the genetic lesion underlying Yaa (Subramanian, S. et al., Proc Natl Acad Sci USA 2006. 103: 9970–9975; Pisitkun, P. et al., Science 2006. 312: 1669–1672). In male mice carrying Yaa, the transcription of several genes within the translocated segment is increased roughly 2-fold. Although the translocated X chromosome segment in Yaa may contain as many as 16 genes, the major candidate gene for causation of the Yaa-associated autoimmune phenotypes has been TLR7. To confirm the role of TLR7 in Yaa-mediated autoimmune phenotypes, we introgressed a targeted disruption of TLR7 (TLR7−) onto B6.Sle1Yaa to produce B6.Sle1YaaTLR7− and examined evidence of disease at 6 and 9 months of age. Our results demonstrate that the upregulation of TLR7 in the B6.Sle1Yaa strain is responsible for splenomegaly, glomerular nephritis and the majority of the cellular abnormalities of B, T and myeloid cells. The upregulation of TLR7 was also responsible for driving the infiltration and activation of leukocytes into the kidney, in which activated T cellswere a primary component. However, the resolution of TLR7 upregulation did not eliminate the enhanced humoral autoimmunity observed in B6.SleYaa, suggesting that additional elements in the translocation may contribute to the disease phenotype.
PMCID: PMC2993003  PMID: 18521959
Autoimmunity; TLR7; genetics; SLE; congenic
23.  Evaluation of C1q genomic region in minority racial groups of lupus 
Genes and immunity  2009;10(5):517-524.
Complement cascade plasma proteins have a complex role in the etiopathogenesis of SLE. Hereditary C1q deficiency has been strongly related to SLE; however, there are very few published SLE studies that evaluate the polymorphisms of the genes encoding for C1q (A, B, and C). In this study, we evaluated 17 single nucleotide polymorphisms (SNPs) across 37 kb of C1QA, B and C in a lupus cohort of peoples of African-American and Hispanic origin. In a case only analysis, significant association at multiple SNPs in the C1QA gene was detected in African-Americans with kidney nephritis (best p=4.91 × 10−6). In addition, C1QA was associated with SLE in African-Americans with a lack of nephritis and accompanying photosensitivity when compared to normal controls (p=6.80 × 10−6). A similar trend was observed in the Hispanic subjects (p=0.003). Quantitative analysis demonstrates that some SNPs in the C1q genes might be correlated with C3 complement levels in an additive model among African-Americans (best p=0.0001). The CIQA gene is associated with subphenotypes of lupus in African-American and Hispanic subjects. Further studies with higher SNP densities in this region and other complement components are necessary to elucidate the complex genetics and phenotypic interactions between complement components and SLE.
PMCID: PMC2769492  PMID: 19440201
24.  Genetic Associations of LYN with Systemic Lupus Erythematosus 
Genes and immunity  2009;10(5):397-403.
We targeted LYN, a src-tyosine kinase involved in B cell activation, in case-control association studies using populations of European American, African American and Korean subjects. Our combined European-derived population, consisting of 2463 independent cases and 3131 unrelated controls, demonstrates significant association with rs6983130 in a female-only analysis with 2254 cases and 2228 controls (p=1.1 × 10−4, OR=0.81 (95% CI: 0.73 – 0.90)). This SNP is located in the 5′ UTR within the first intron near the transcription initiation site of LYN. Additional SNPs upstream of the first exon also show weak and sporadic association in subsets of the total European American population. Multivariate logistic regression analysis implicates rs6983130 as a protective factor for SLE susceptibility when anti-dsDNA, anti-chromatin, anti-52 kDa Ro or anti-Sm autoantibody status were used as covariates. Subset analysis of the European American female cases by ACR classification criteria reveals a reduction in the risk of hematologic disorder with rs6983130 compared to cases without hematologic disorders (p=1.5 × 10−3, OR=0.75 (95% C.I.=0.62-0.89)). None of the 90 SNPs tested demonstrate significant association with SLE in the African American or Korean populations. These results support an association of LYN with European-derived individuals with SLE, especially within autoantibody or clinical subsets.
PMCID: PMC2750001  PMID: 19369946
systemic lupus erythematosus; association; LYN; SNP
25.  The lupus susceptibility gene kallikrein down-modulates antibody-mediated glomerulonephritis 
Genes and immunity  2009;10(5):503-508.
Sle3 is an NZM2410/NZW-derived lupus susceptibility interval on murine chromosome 7, that is associated with spontaneous lupus nephritis, and also anti-GBM induced glomerulonephritis. The tissue kallikrein gene cluster is located within the Sle3 interval and constitutes potential candidate genes for this locus. We have recently reported that renal kallikrein expression was up-regulated by anti-GBM antibody challenge in a strain-specific manner and that it was significantly under-expressed in the anti-GBM sensitive strains, including B6.Sle3. Further sequencing and functional studies reported previously provided evidence that kallikreins could constitute disease genes in lupus. In the present report, we have used an adenoviral vector to deliver the klk1 gene to B6.Sle3 congenics to directly test if kallikreins might have a protective effect against anti-GBM induced nephritis. Our data shows that klk1 gene delivery ameliorated anti-GBM induced nephritis in B6.Sle3 congenics. Taken together with previous studies, these findings indicate that kallikreins play an important protective role in autoantibody-initiated glomerulonephritis, and could constitute potential candidate genes for anti-GBM induced and spontaneous lupus nephritis.
PMCID: PMC2742360  PMID: 19262577
Kallikrein; anti-GBM; glomerulonephritis; adenovirus; lupus

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