DNA corona on nanoparticles leads to an enhanced immunostimulatory effect with implications for autoimmune diseases.

Autoimmune and inflammatory diseases are highly complex, limiting treatment and the development of new therapies. Recent work has shown that cell-free DNA bound to biological microparticles is linked to systemic lupus erythematosus, a prototypic autoimmune disease. However, the heterogeneity and technical challenges associated with the study of biological particles have hindered a mechanistic understanding of their role. Our goal was to develop a well-controlled DNA-particle model system to understand how DNA-particle complexes affect cells. We first characterized the adsorption of DNA on the surface of polystyrene nanoparticles (200 nm and 2 µm) using transmission electron microscopy, dynamic light scattering, and colorimetric DNA concentration assays. We found that DNA adsorbed on the surface of nanoparticles was resistant to degradation by DNase 1. Macrophage cells incubated with the DNA-nanoparticle complexes had increased production of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We probed two intracellular DNA sensing pathways, toll-like receptor 9 (TLR9) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), to determine how cells sense the DNA-nanoparticle complexes. We found that the cGAS-STING pathway is the primary route for the interaction between DNA-nanoparticles and macrophages. These studies provide a molecular and cellular-level understanding of DNA-nanoparticle-macrophage interactions. In addition, this work provides the mechanistic information necessary for future in vivo experiments to elucidate the role of DNA-particle interactions in autoimmune diseases, providing a unique experimental framework to develop novel therapeutic approaches.

In patients at high risk for RA, abatacept reduced inflammation and RA development at 6 mo.

SOURCE CITATION: Rech J, Tascilar K, Hagen M, et al. Abatacept inhibits inflammation and onset of rheumatoid arthritis in individuals at high risk (ARIAA): a randomised, international, multicentre, double-blind, placebo-controlled trial. Lancet. 2024;403:850-859. 38364841.

The role of DNA in the pathogenesis of SLE: DNA as a molecular chameleon.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterised by antibodies to DNA (anti-DNA) and other nuclear macromolecules. Anti-DNA antibodies are markers for classification and disease activity and promote pathogenesis by forming immune complexes that deposit in the tissue or stimulate cytokine production. Studies on the antibody response to DNA have focused primarily on a conformation of DNA known as B-DNA, the classic right-handed double helix. Among other conformations of DNA, Z-DNA is a left-handed helix with a zig-zag backbone; hence, the term Z-DNA. Z-DNA formation is favoured by certain base sequences, with the energetically unfavourable flip from B-DNA to Z-DNA dependent on conditions. Z-DNA differs from B-DNA in its immunogenicity in animal models. Furthermore, anti-Z-DNA antibodies, but not anti-B-DNA antibodies, can be present in otherwise healthy individuals. In SLE, antibodies to Z-DNA can occur in association with antibodies to B-DNA as a cross-reactive response, rising and falling together. While formed transiently in chromosomal DNA, Z-DNA is stably present in bacterial biofilms; biofilms can provide protection against antibiotics and other challenges including elements of host defence. The high GC content of certain bacterial DNA also favours Z-DNA formation as do DNA-binding proteins of bacterial or host origin. Together, these findings suggest that sources of Z-DNA can enhance the immunogenicity of DNA and, in SLE, stimulate the production of cross-reactive antibodies that bind both B-DNA and Z-DNA. As such, DNA can act as a molecular chameleon that, when stabilised in the Z-DNA conformation, can drive autoimmunity.

2023 International Consensus Guidance for the use of Tripterygium Wilfordii Hook F in the treatment of active rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disorder that affects the joints and produces pain, swelling, and stiffness. It has a lifetime prevalence of up to 1% worldwide. An extract of Tripterygium wilfordii Hook F (TwHF), a member of the Celastraceae herbal family widely available in south China, has been used for treatment of RA since 1960s. METHODS: The current consensus practice guidance (CPG) aims to offer guidance on the application of TwHF in the clinical management of active RA. The CPG followed World Health Organisation (WHO)'s recommended process, carried out three systematic reviews to synthesize data from 19 randomised controlled trials (RCT) involving 1795 participants. We utilized Grading of Recommendations, Assessment, Development and Evaluation (GRADE) to evaluate certainty of evidence and derive recommendations. We rigorously followed The Appraisal of Guidelines for Research and Evaluation II (AGREE II) as conduct guides to minimise bias and promote transparency. RESULTS: There was no obvious difference between TwHF monotherapy and methotrexate (MTX) monotherapy on ACR20 (RCT = 2, N = 390, RR = 1.06, 95%CI 0.90-1.26, moderate certainty), ACR50 (RCT = 3, N = 419, RR = 1.03, 95%CI 0.80-1.34, moderate certainty), ACR70 (RCT = 2, N = 390, RR = 1.12, 95%CI 0.69-1.79, low certainty). TwHF monotherapy may be better than salicylazosulfapyridine monotherapy on ACR20 and the effect may be similar on ACR50 and ACR70. Seven RCTs compared MTX combined with TwHF versus MTX monotherapy, and the meta-analysis results favoured combination therapy group on ACR20 (RCT = 3, N = 470, RR = 1.44, 95%CI 1.28-1.62, moderate certainty), ACR50 (RCT = 4, N = 500, RR = 1.88, 95%CI 1.56-2.28, moderate certainty) and ACR70 (RCT = 2, N = 390, RR = 2.12, 95%CI 1.40-3.19, low certainty). We found no obvious difference between groups on critical safety outcomes, including infection (RCT = 3, N = 493, RR = 1.37, 95%CI 0.84-2.23), liver dysfunction (RCT = 5, N = 643, RR = 1.14, 95%CI 0.71-1.85), renal damage (RCT = 3, N = 450, RR = 2.20, 95%CI 0.50-9.72). CONCLUSION: Upon full review of the evidence, the guidance panel reached consensus on recommendations for the use of TwHF in people with active RA, either as monotherapy or as combination therapy with MTX.

Patient and Physician Perspectives of Systemic Lupus Erythematosus Flare: A Qualitative Study.

OBJECTIVE: Systemic lupus erythematosus (SLE) flares are associated with increased damage and decreased health-related quality of life. We hypothesized that there is discordance between physicians' and patients' views of SLE flare. In this study, we aimed to explore patient and physician descriptions of SLE flares. METHODS: We conducted a qualitative descriptive study using in-depth interviews with a purposeful sample of patients with SLE (who met 1997 American College of Rheumatology or Systemic Lupus International Collaborating Clinics criteria) and practicing rheumatologists. Interviews were audio-recorded, transcribed, and analyzed using applied thematic analysis. RESULTS: Forty-two patient participants with SLE, representing a range of SLE activity, completed interviews. The majority described flare symptoms as joint pain, fatigue, and skin issues lasting several days. Few included objective signs or laboratory measures, when available, as features of flare. We interviewed 13 rheumatologists from 10 academic and 3 community settings. The majority defined flare as increased or worsening SLE disease activity, with slightly more than half requiring objective findings. Around half of the rheumatologists included fatigue, pain, or other patient-reported symptoms. CONCLUSION: Patients and physicians described flare differently. Participants with SLE perceived flares as several days of fatigue, pain, and skin issues. Providers defined flares as periods of increased clinical SLE activity. Our findings suggest the current definition of flare may be insufficient to integrate both perceptions. Further study is needed to understand the pathophysiology of patient flares and the best way to incorporate patients' perspectives into clinical assessments.

The Type 1 & 2 systemic lupus erythematosus model: Perspectives of people living with systemic lupus erythematosus.

OBJECTIVE: In the new Type 1 & 2 model for systemic lupus erythematosus (SLE), Type 1 SLE represents classic inflammatory manifestations, such as arthritis, while Type 2 SLE encompasses symptoms such as pain and fatigue where the relationship to inflammation is less clear. The objective of this study was to interview individuals living with SLE to determine the content and face validity of the Type 1 & 2 SLE model. METHODS: We conducted a qualitative study using semi-structured interviews with a purposeful sample of participants who met classification criteria for SLE. Participants were asked to describe their experiences with Type 1 & 2 SLE symptoms and treatments, and they indicated if and how their personal experiences aligned with the Type 1 & 2 SLE model. All interviews were audio-recorded and transcribed; applied thematic analysis identified the most frequent and salient themes. RESULTS: We interviewed 42 participants with SLE. Type 2 SLE symptoms, such as pain and fatigue, were very common, with almost all participants experiencing some Type 2 symptoms at some point during their disease course. Participants described Type 1 SLE symptoms as being acute flares and life-threatening and Type 2 SLE symptoms as "everyday lupus" that affected their daily lives and were a dominant part of their SLE disease experience. Most participants stated they want their rheumatologists to discuss Type 2 symptoms during clinical appointments in order to address their full symptom experience. CONCLUSION: We demonstrated content and face validity of the Type 1 & 2 SLE model with people living with SLE. Participants in our study largely understood the model and felt it accurately reflected their experience living with SLE. Type 2 SLE symptoms are very common in individuals with SLE and impact patients' quality of life. Using the model to address Type 2 SLE symptoms allows the rheumatologist to incorporate the patient's perspective and provide patient-centered care.

In relapsing AAV with rituximab-induced remission, maintenance rituximab vs. azathioprine reduced relapse at ≥32 mo.

SOURCE CITATION: Smith RM, Jones RB, Specks U, et al; RITAZAREM co-investigators. Rituximab versus azathioprine for maintenance of remission for patients with ANCA-associated vasculitis and relapsing disease: an international randomised controlled trial. Ann Rheum Dis. 2023;82:937-944. 36958796.

In glucocorticoid-dependent polymyalgia rheumatica, tocilizumab improved a composite clinical outcome at 24 wk.

SOURCE CITATION: Devauchelle-Pensec V, Carvajal-Alegria G, Dernis E, et al. Effect of tocilizumab on disease activity in patients with active polymyalgia rheumatica receiving glucocorticoid therapy: a randomized clinical trial. JAMA. 2022;328:1053-62. 36125471.

The Binding Properties of Antibodies to Z-DNA in the Sera of Normal Healthy Subjects.

Antibodies to DNA are a diverse set of antibodies that bind sites on DNA, a polymeric macromolecule that displays various conformations. In a previous study, we showed that sera of normal healthy subjects (NHS) contain IgG antibodies to Z-DNA, a left-handed helix with a zig-zig backbone. Recent studies have demonstrated the presence of Z-DNA in bacterial biofilms, suggesting a source of this conformation to induce responses. To characterize further antibodies to Z-DNA, we used an ELISA assay with brominated poly(dGdC) as a source of Z-DNA and determined the isotype of these antibodies and their binding properties. Results of these studies indicate that NHS sera contain IgM and IgA as well as IgG anti-Z-DNA antibodies. As shown by the effects of ionic strength in association and dissociation assays, the anti-Z-DNA antibodies bind primarily by electrostatic interactions; this type of binding differs from that of induced anti-Z-DNA antibodies from immunized animals which bind by non-ionic interactions. Furthermore, urea caused dissociation of NHS anti-Z-DNA at molar concentrations much lower than those for the induced antibodies. These studies also showed IgA anti-Z-DNA antibodies in fecal water. Together, these studies demonstrate that antibodies to Z-DNA occur commonly in normal immunity and may arise as a response to Z-DNA of bacterial origin.

The expression of antibodies to Z-DNA in the blood of patients with systemic lupus erythematosus: Relationship to autoantibodies to B-DNA.

To explore the antibody response to Z-DNA, a DNA conformation with a zig-zag structure, blood of patients with systemic lupus erythematosus (SLE) and otherwise healthy individuals (NHS) were assayed by ELISA using brominated poly(dGdC), a synthetic Z-DNA antigen. These studies showed that SLE patients commonly express antibodies to Z-DNA; NHS also had binding in this assay. In SLE blood, levels of antibodies to Z-DNA were related to those to B-DNA using calf thymus DNA as a source of B-DNA; cross-reactivity was demonstrated by adsorption experiments using DNA cellulose. As shown by dissociation assays, antibody binding of SLE anti-Z-DNA is sensitive to the effects of ionic strength, suggesting electrostatic binding. Since Z-DNA structure can be found in bacterial DNA as well as bacterial biofilms, these findings suggest that, in SLE, anti-DNA antibody responses can result from stimulation by DNA of bacterial origin, with cross-reactivity leading to autoreactivity.

Annals of the Rheumatic Diseases collection on autoantibodies in the rheumatic diseases: new insights into pathogenesis and the development of novel biomarkers.

The rheumatic diseases are a diverse group of conditions that can display autoantibody production, functional immune disturbances and systemic disease manifestations. These autoantibodies can serve as markers for classification, diagnosis, prognosis and disease activity. Among specificities prominently expressed by patients, those directed to nuclear antigens (antinuclear antibodies or ANAs) are markers for specific rheumatic diseases. ANAs can bind to DNA, RNA and complexes of proteins with nucleic acids. Other autoantibodies expressed in the rheumatic diseases are directed to proteins, including IgG, post-translational modifications of proteins and soluble mediators such as cytokines. While autoantibodies have been investigated for over 50 years, recent studies published in the Annals of the Rheumatic Diseases (ARD) have provided an exciting perspective on the mechanisms of autoantibody production and the power of new technologies to identify novel autoantibody targets to elucidate aetiology and underpin patient evaluation. Furthermore, in-depth serological studies have demonstrated a phenomenon known as clustering; clustering defines sets of autoantibodies that are commonly expressed together in patients with a given rheumatic disease. Other research reported in ARD has used B cell phenotyping and genotypic analysis to subtype patients, and has explored the relationship of autoantibodies, complement activation and patterns of gene expression as exemplified by the interferon gene signature. Together, these studies provide important new insights into disease mechanisms as well as actionable information to facilitate personalised patient care.

Anti-RNP antibodies are associated with the interferon gene signature but not decreased complement levels in SLE.

OBJECTIVES: The goals of these studies were to elucidate the inter-relationships of specific anti-nuclear antibody (ANA), complement, and the interferon gene signature (IGS) in the pathogenesis of systemic lupus erythematosus (SLE). METHODS: Data from the Illuminate trials were analysed for antibodies to dsDNA as well as RNA-binding proteins (RBP), levels of C3, C4 and various IGS. Statistical hypothesis testing, linear regression analyses and classification and regression trees analysis were employed to assess relationships between the laboratory features of SLE. RESULTS: Inter-relationships of ANAs, complement and the IGS differed between patients of African Ancestry (AA) and European Ancestry (EA); anti-RNP and multiple autoantibodies were more common in AA patients and, although both related to the presence of the IGS, relationships between autoantibodies and complement differed. Whereas, anti-dsDNA had an inverse relationship to C3 and C4, levels of anti-RNP were not related to these markers. The IGS was only correlated with anti-dsDNA in EA SLE and complement was more correlated to the IGS in AA SLE. Finally, autoantibodies occurred in the presence and absence of the IGS, whereas the IGS was infrequent in anti-dsDNA/anti-RBP-negative SLE patients. CONCLUSION: There is a complex relationship between autoantibodies and the IGS, with anti-RNP associated in AA and both anti-dsDNA and RNP associated in EA. Moreover, there was a difference in the relationship between anti-dsDNA, but not anti-RBP, with complement levels. The lack of a relationship of anti-RNP with C3 and C4 suggests that anti-RNP immune complexes (ICs) may drive the IGS without complement fixation, whereas anti-dsDNA ICs involve complement consumption.

Metabolomics analysis identifies a lipidomic profile in treatment-naïve juvenile dermatomyositis patients vs healthy control subjects.

OBJECTIVES: To perform an exploratory study to identify a JDM serum metabolic profile that differs from healthy controls (HCs) and responds to immunosuppressive treatment. METHODS: Blood was collected from 9 HCs and 10 patients diagnosed with probable (n = 4) or definite (n = 6) JDM based on the criteria of Bohan and Peter for myositis, with 7 of the 10 providing longitudinal samples following initiation of treatment; these patients comprised the treatment-naïve cohort. Sera underwent mass spectroscopy-based measurements of targeted metabolic intermediates, including 15 amino acids, 45 acylcarnitines (ACs), 15 ceramides and 29 sphingomyelins. Principal components analysis reduced metabolites into smaller sets of factors each comprised of correlated metabolic intermediates. Factor scores and metabolite concentrations were compared with HCs using two-sample t-tests while treatment effects were evaluated using paired t-tests. RESULTS: Of eight principal components analysis-derived metabolite factors (one AC, two amino acids, three sphingosine and two ceramide), two were significantly associated with JDM: one AC factor containing mostly long-chain ACs (P = 0.049) and one ceramide factor (P < 0.01). For 12 individual ACs, mostly long chain, and three ceramides, concentrations were significantly greater for JDM than HCs. Factors based on these individual metabolites showed decreasing scores with treatment (P = 0.03 and P < 0.01, respectively). CONCLUSION: While additional validation is needed, these lipids have potential as JDM serum diagnostic and/or treatment biomarkers. Additionally, the significant association of long-chain ACs and ceramides with JDM offers insights regarding pathogenesis, implicating dysregulation of mitochondrial fatty acid ß-oxidation.

The use of patient-reported outcome measures to classify type 1 and 2 systemic lupus erythematosus activity.

OBJECTIVE: We developed a model that categorizes systemic lupus erythematosus (SLE) activity into two dimensions: Type 1 SLE consists of inflammatory activity, including arthritis, nephritis, and rashes; Type 2 SLE includes fatigue, myalgia, mood disturbance, and cognitive dysfunction. Patient-reported outcome (PRO) measures have received attention as a way to capture symptomatology of SLE. The objective of this study was to explore the use of existing PRO measures to classify Type 1 and 2 SLE activity. METHODS: Systemic lupus erythematosus patients completed three questionnaires: Systemic Lupus Activity Questionnaire (SLAQ), Polysymptomatic Distress Scale (PSD), and Patient Health Questionnaire (PHQ-2). SLE Disease Activity Index (SLEDAI) and physician global assessments (PGA; 0-3) for Type 1 and Type 2 activity were also recorded. High Type 1 SLE activity was defined as cSLEDAI ≥4 (scored without labs), SLEDAI ≥6, active nephritis, or Type 1 PGA ≥1.0. High Type 2 SLE activity was defined as Type 2 PGA ≥1.0. Patients with both high Type 1 and 2 activity were defined as Mixed SLE, and patients with low Type 1 and 2 activity were defined as Minimal SLE. Data were reduced with a factor analysis. Using a reduced set of 13 variables, multinomial logistic regression models estimated the probability of Minimal, Type 1, Type 2, and Mixed SLE classification. RESULTS: The study included 208 patients with SLE. The model accurately predicted the clinician-based Type 1 and 2 SLE classification in 63% of patients; 73% of patients had their Type 1 SLE activity accurately predicted; and 83% had their Type 2 SLE activity accurately predicted. Performance varied by group: 87% of Minimal patients were correctly predicted to be in the Minimal SLE group, yet only about one-third of patients in the Type 1 group were correctly predicted to be in the Type 1 group. CONCLUSIONS: Our findings indicate Type 2 SLE activity can be identified by patient-reported data. The use of PROs was not as accurate at predicting Type 1 activity. These findings highlight the challenges of using PROs to categorize and classify SLE symptoms since some manifestations of Type 1 activity (e.g., nephritis) may be essentially clinically silent while other Type 1 manifestations may cause severe symptoms.

Some disease-modifying osteoarthritis drugs make small improvements in knee and hip osteoarthritis.

SOURCE CITATION: Yang W, Sun C, He SQ, et al. The efficacy and safety of disease-modifying osteoarthritis drugs for knee and hip osteoarthritis-a systematic review and network meta-analysis. J Gen Intern Med. 2021;36:2085-93. 33846938.

The Interaction of Anti-DNA Antibodies with DNA: Evidence for Unconventional Binding Mechanisms.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus, a prototypic autoimmune disease. These antibodies bind to conserved sites on single-stranded and double-stranded DNA and display variable region somatic mutations consistent with antigen selection. Nevertheless, the interaction of anti-DNA with DNA has unconventional features. Anti-DNA antibodies bind by a mechanism called monogamous bivalency, in which stable interaction requires contact of both Fab sites with determinants on the same extended DNA molecule; the size of this DNA can be hundreds to thousands of bases, especially in solid phase assays. This binding also requires the presence of the Fc portion of IgG, a binding mechanism known as Fc-dependent monogamous bivalency. As shown by the effects of ionic strength in association and dissociation assays, anti-DNA binding is primarily electrostatic. Like anti-DNA autoantibodies, anti-DNA antibodies that bind specifically to non-conserved sites on bacterial DNA, a type of anti-DNA found in otherwise healthy individuals, also interact by monogamous bivalency. The unconventional features of anti-DNA antibodies may reflect the highly charged and polymeric nature of DNA and the need for molecular rearrangements to facilitate monogamous bivalency; the Fc portion contributes to binding in an as yet unknown way.

The interaction of anti-DNA antibodies with DNA antigen: Evidence for hysteresis for high avidity binding.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus. Previous studies have indicated that the phosphodiester backbone is the main antigenic target, with electrostatic interactions important for high avidity. To define further these interactions, the effects of ionic strength on anti-DNA binding of SLE plasmas were assessed in association and dissociation assays by ELISA. As these studies demonstrated, increasing ionic strength to a concentration of 1000 mM NaCl reduced antibody binding although the extent of the reduction varied among samples. In dissociation assays, differences among plasmas were also observed. For one of the plasmas, binding to DNA displayed resistance to dissociation by increasing ionic strength even though these concentrations limited binding in association assays. Time course studies showed a gradual change in binding interactions. These studies indicate that anti-DNA binding can involve both electrostatic and non-electrostatic interactions, with binding in some plasmas showing evidence of hysteresis.

The Binding of Monoclonal and Polyclonal Anti-Z-DNA Antibodies to DNA of Various Species Origin.

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichiacoli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.

The binding of SLE autoantibodies to mitochondria.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by immune complexes. Because these complexes contain mitochondrial components, we assessed the presence of antibodies to whole mitochondria (wMITO) using an ELISA in which mitochondria from mouse liver are bound to microtiter plates pre-coated with poly-l-lysine. Studies with this ELISA demonstrated that SLE plasmas contain abundant anti-wMITO activity. While digestion with DNase 1 did not affect anti-wMITO activity, adsorption of plasma on DNA affinity columns could reduce binding activity. Assay for anti-mitochondrial antibodies (AMA) by immunofluorescence and an ELISA with the M2 antigen (2-oxo-acid dehydrogenase protein complex) showed a low frequency of positivity, indicating that AMA and anti-wMITO are distinct specificities. In the study of 204 patients with SLE, the levels of anti-wMITO were higher in active SLE and correlated with levels of anti-DNA. These findings suggest that anti-wMITO can form immune complexes with mitochondria which may drive pathogenesis.

Evolving story of autoantibodies in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by antinuclear antibody (ANA) production. ANAs bind to DNA, RNA and complexes of proteins and nucleic acids and are important markers for diagnosis and activity. According to current models, ANAs originate from antigen-driven processes; nevertheless, antibody responses to both DNA and RNA binding proteins display features unexpected in terms of current paradigms for antigenicity. These differences may reflect disturbances in both B and T cells critical for autoreactivity. Clinically, ANA testing has new uses for determining classification as well as assessing eligibility for clinical trials. Studies of patients with established disease show frequent seronegativity. In this setting, seronegativity may indicate a stage of disease called post-autoimmunity in which the natural history of disease or effects of immunosuppressive therapies modifies responses. The new uses of ANA testing highlight the importance of understanding autoantigenicity and developing sensitive and informative assays for clinical assessments.