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Objective Many Native American (NA) patients with systemic lupus erythematosus (SLE) do not exhibit the classical SLE autoantibody profiles of European American (EA) and African American (AA) patients with SLE. The poorer SLE disease outcomes noted in NA patients highlights a need for more equitable diagnostic and prognostic tools for NA patients with SLE. The objective was to identify informative autoantibody profiles for NA, AA, and EA patients with SLE using an expanded set of autoantigens. Methods Sera from 49 NA, 49 AA, and 49 EA age-, sex-, and antinuclear autoantibody titer-matched patients with SLE who met the American College of Rheumatology classification criteria and 10 ethnicity-, sex-, and age-matched controls were tested for autoantibody reactivity by autoantigen microarrays. Autoantibodies that were significantly elevated in patients with SLE compared with ethnicity-specific controls were selected for hierarchical clustering. Differences in clinical criteria between patient clusters were determined by Fisher's exact test and corrected for multiple comparisons. Results NA, AA, and EA patients with SLE each had a cluster distinguished by higher levels of anti-Ro52 and another cluster distinguished by nucleic acid-specific autoantibodies. Additional clusters were distinguished in NA patients by elevated extracellular matrix autoantibodies and were distinguished in AA patients by elevated Sm/RNP autoantibody and elevated nucleolin/histone autoantibody. Two EA patient clusters with similar nucleic acid- and Ro52-specific autoantibodies were distinguished by either high or low histone 2A reactivity. Renal manifestations trended higher in the NA Ro52 cluster and were significantly enriched in the AA nucleolin/histone cluster. The AA nucleolin/histone cluster and EA H2A cluster had higher disease activity. Conclusion Expanded autoantibody profiles can identify informative subsets of patients with SLE.

Progression from health to a classified autoimmune disease is an evolving process that can happen rapidly in some diseases, but usually takes years to develop. Specific immune alterations predate pathogenic autoimmunity and can be used as disease biomarkers to identify high-risk individuals for prevention studies applied in the pre-clinical state. Here we discuss recent findings that illuminate specific immune pathways that are altered in the earliest phases of pre-clinical autoimmunity as well as those mediators more closely associated with later clinically apparent and classified disease onset.

Systemic lupus erythematosus (SLE) flares elicit progressive organ damage, leading to disability and early mortality. This study evaluated clinical and immunologic factors associated with impending flare in the Biomarkers of Lupus Disease study. Autoantibodies and 32 soluble mediators were measured by multiplex assays, immune pathway activation by gene expression module scores, and immune cell subset frequencies and activation states by flow cytometry. After providing baseline samples, participants received transient steroids to suppress disease and were followed until flare. Flare occurred early (within 60 days of baseline) in 21 participants and late (90-165 days) in 13. At baseline, compared to the late flare group, the early flare group had differential gene expression in monocyte, T cell, interferon, and inflammation modules, as well as significantly higher frequencies of activated (aCD11b

ObjectiveTo address heterogeneity complicating primary SS (pSS) clinical trials, research and care by characterizing and clustering patients by their molecular phenotypes.MethodspSS patients met American–European Consensus Group classification criteria and had at least one systemic manifestation and stimulated salivary flow of ⩾0.1 ml/min. Correlated transcriptional modules were derived from gene expression microarray data from blood (n = 47 with appropriate samples). Patients were clustered based on this molecular information using an unbiased random forest modelling approach. In addition, multiplex, bead-based assays and ELISAs were used to assess 30 serum cytokines, chemokines and soluble receptors. Eleven autoantibodies, including anti-Ro/SSA and anti-La/SSB, were measured by Bio-Rad Bioplex 2200.ResultsTranscriptional modules distinguished three clusters of pSS patients. Cluster 1 showed no significant elevation of IFN or inflammation modules. Cluster 2 showed strong IFN and inflammation modular network signatures, as well as high plasma protein levels of IP-10/CXCL10, MIG/CXCL9, BLyS (BAFF) and LIGHT. Cluster 3 samples exhibited moderately elevated IFN modules, but with suppressed inflammatory modules, increased IP-10/CXCL10 and B cell–attracting chemokine 1/CXCL13 and trends toward increased MIG/CXCL9, IL-1α, and IL-21. Anti-Ro/SSA and anti-La/SSB were present in all three clusters.ConclusionMolecular profiles encompassing IFN, inflammation and other signatures can be used to separate patients with pSS into distinct clusters. In the future, such profiles may inform patient selection for clinical trials and guide treatment decisions.

Systemic lupus erythematosus (SLE) flares elicit progressive organ damage, leading to disability and early mortality. This study evaluated clinical and immunologic factors associated with impending flare in the Biomarkers of Lupus Disease study. Autoantibodies and 32 soluble mediators were measured by multiplex assays, immune pathway activation by gene expression module scores, and immune cell subset frequencies and activation states by flow cytometry. After providing baseline samples, participants received transient steroids to suppress disease and were followed until flare. Flare occurred early (within 60 days of baseline) in 21 participants and late (90–165 days) in 13. At baseline, compared to the late flare group, the early flare group had differential gene expression in monocyte, T cell, interferon, and inflammation modules, as well as significantly higher frequencies of activated (aCD11b+) neutrophils and monocytes, and activated (CD86hi) naïve B cells. Random forest models showed three subgroups of early flare patients, distinguished by greater baseline frequencies of aCD11b+ monocytes, or CD86hi naïve B cells, or both. Increases in these cell populations were the most accurate biomarkers for early flare in this study. These results suggest that SLE flares may arise from an overlapping spectrum of lymphoid and myeloid mechanisms in different patients.

Objective Incomplete lupus erythematosus (ILE) involves clinical and/or serologic manifestations consistent with but insufficient for systemic lupus erythematosus (SLE) classification. Because the nature of ILE is poorly understood and no treatment recommendations exist, we examined the clinical manifestations, medication history, and immunologic features in a diverse collection of ILE and SLE patients. Methods Medical records of subjects enrolled in the Lupus Family Registry and Repository were reviewed for medication history and American College of Rheumatology (ACR) classification criteria to identify ILE patients (3 ACR criteria; n = 440) and SLE patients (≥4 ACR criteria; n = 3,397). Participants completed the Connective Tissue Disease Screening Questionnaire. Anticardiolipin and plasma B lymphocyte stimulator (BLyS) were measured by enzyme-linked immunosorbent assay, antinuclear antibodies (ANAs) by indirect immunofluorescence, and 13 autoantibodies by bead-based assays. Results On average, ILE patients were older than SLE patients (46.2 years versus 42.0 years; P < 0.0001), and fewer ILE patients were African American (23.9% versus 32.2%; P < 0.001). ILE patients exhibited fewer autoantibody specificities than SLE patients (1.3 versus 2.6; P < 0.0001) and were less likely to have ANA titers ≥1:1,080 (10.5% versus 19.5%; P < 0.0001). BLyS levels were intermediate in ILE patients (controls < ILE; P = 0.016; ILE < SLE; P = 0.008). Pericarditis, renal, or neurologic manifestations occurred in 12.5% of ILE patients and were associated with non–European American race/ethnicity (P = 0.012). Hydroxychloroquine use increased over time, but was less frequent in ILE than SLE patients (65.2% versus 83.1%; P < 0.0001). Conclusion Although usually characterized by milder symptoms, ILE manifestations may require immunomodulatory treatments. Longitudinal studies are necessary to understand how ILE affects organ damage and future SLE risk, and to delineate molecular pathways unique to ILE.

Infections remain a significant source of morbidity and mortality in systemic lupus erythematosus (SLE). Both the underlying disease process and therapeutic immunosuppression can increase susceptibility to a wide variety of pathogens while influencing the clinical course of infections. Because of the atypical presentation of many infections and the varied array of baseline symptoms, distinguishing an acute infection from a lupus flare is a common clinical challenge in treating SLE patients. This challenge is compounded by the need to suppress the immune system enough to prevent autoimmune complications without making patients overly vulnerable to infection. This chapter reviews the impact of infections on lupus morbidity and mortality, risk factors for infection, the clinical spectrum of infections in lupus, proposed mechanisms of infection susceptibility, and biomarkers or other factors that may assist the clinician when differentiating active infection from an SLE disease flare.

Background Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease oftentimes characterised by a waxing and waning disease course. However, mechanisms of disease flare remain elusive. Objectives This study examined relationships between SLE disease activity, immune pathways, and serologic evidence of viral exposures and reactivation within molecular subsets of SLE patients. Methods Serial or single samples of plasma, serum and RNA (n=290) were collected from 184 adult SLE patients who met ACR classification and cohort-matched controls (n=49). Disease activity was assessed by modified SELENA-SLEDAI. Immune pathways were evaluated by modular transcriptional analysis of Illumina Beadchip Microarray gene expression data, as well as by plasma soluble mediators (n=32) by multiplex, bead-based assay and ELISAs. This information was used to subset patients into seven molecular-defined categories by random forest modelling. Viral seropositivity and antibody concentrations were detected by ELISA for antibodies against EBV-Viral Capsid Antigen (VCA) (IgG and IgA), EBV-Early Antigen (EA) (IgG), Cytomegalovirus (CMV) (IgG), and Herpes Simplex Virus (HSV-1) (IgG). Results Serologic evidence of EBV reactivation was more common in SLE patients compared to controls, as measured by antibodies against EBV-EA [IgG] (40% vs 13%; OR=4.57, p=0.0006) or EBV-VCA [IgA] (36% vs 17%; OR=2.70, p=0.019). No differences were noted in CMV or HSV1 seropositivity rates between patients and controls. IgG responses against EBV-VCA were nearly universal among these adult patients and controls; however, concentrations of EBV-VCA IgG were higher in SLE patients compared to controls (ISR=4.44 vs 3.52; p=0.0021), as were EBV-VCA IgA and EBV-EA IgG antibody responses. In cross sectional analysis, SLE patients with higher disease activity (SLEDAI ≥6; n=126) had higher concentrations of EBV-EA IgG than patients with lower (n=166) disease activity (ISR=0.822 vs 0.540; p=0.033). SLE patients with serologic evidence of EBV reactivation by EA IgG responses had higher levels of interferon associated molecules, IP10 (p=3.4×10–14), BLyS (5.5×10–5), and IL-10 (p=0.00013). HSV1 IgG positive SLE patients also showed higher levels of IP10 (2.2×10–7). Antibody responses toward EBV-EA were enriched in molecularly defined patient clusters with higher expression levels of interferon and inflammatory modules, as well as with interferon and inflammatory soluble mediators. Patients within these clusters were also more likely to have major organ involvement, such as renal or neurologic disease. Conclusions Serologic evidence of EBV reactivation is more common in SLE patients compared to healthy controls. EBV-EA IgG responses are elevated in SLE patients with active disease and correspond with increases in interferon-associated mediators. This study provides serologic evidence suggesting a possible role for viral reactivation in SLE disease activity. Acknowledgements This work was supported in part by grants from the National Institutes of Health: U19AI082714, U01AI101934, U54GM104938, P30AR053483, R01AR072401. Disclosure of Interest None declared

Background Systemic lupus erythematosus (SLE) is characterised by remarkable clinical and pathophysiological diversity, hindering diagnosis, treatment and treatment development. Subsetting of patients based upon clinical presentations alone has not identified homogeneous groups of patients best treated with a directed therapeutic. Objectives To cluster SLE patients into more homogeneous subsets with common molecular pathway signatures, and to assess the clinical, therapeutic, and demographic features enriched in each cluster. Methods Serial or single plasma, serum and RNA samples (n=290) were collected from 198 SLE patients who met ACR classification. Disease activity was assessed by modified SELENA-SLEDAI at an average of 17 visits per patient. Transcriptional co-expression signature module scores were calculated from Illumina Beadchip Microarray gene expression data for 29 immune pathway related modules. Plasma soluble mediators (n=23) and 12 antinuclear autoantibodies (anti-dsDNA, chromatin, ribosomal P, Sm, Sm, SmRNP, RNP, Ro/SSA, La/SSB, centromere B, Scl-70 and Jo-1) were assessed by multiplex bead-based assay and ELISA. Spearman correlations were used for univariate and multivariate analysis using R. Patients were clustered on module signature scores and soluble mediators using random forest and tSNE. Results SLEDAI scores strongly correlated with interferon modules and were modestly correlated with plasmablast and select cell cycle signatures in this adult lupus collection. SLEDAI scores also correlated with soluble levels of IFNa, IL21, IL1a, IL17A, IP10 and MIG. Random forest defined seven clusters of SLE patients with unique molecular phenotypes based upon gene co-expression module signatures and soluble mediators. Inflammation and interferon (IFN) signatures were elevated in Clusters 1 (moderately) and 4, with decreased T cell signatures in Cluster 4. The other clusters had lower IFN and inflammation signatures, but differed in their monocyte, plasmablast and T cell signatures. Clusters 1 and 4 had the highest SLEDAI scores, with high rates of anti-dsDNA, low complement, proteinuria and hematuria; these features were also prominent in Cluster 3, which lacked the IFN and inflammation signatures. Cluster 6 had the highest plasmablast module score, highest IL1a levels, and SLEDAI scored rashes, but only moderate IFN and inflammation module scores. Cluster 2 had higher rates of SLEDAI scored alopecia, a slightly elevated inflammation signature, but lower interferon signature and lower soluble mediator levels. Conclusions SLE subsets can be distinguished by a range of molecular profiles encompassing IFN, T cell, neutrophil, plasmablast, and inflammation co-expression signatures, as well as soluble mediators that vary with disease activity. Prospective longitudinal studies of these molecular profiles may inform clinical trial design and personalised disease management. Acknowledgements This work was supported in part by grants from the National Institutes of Health: U19AI082714, U01AI101934, U54GM104938, and P30AR053483. Disclosure of Interest None declared

Objective: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with varied morbidity and mortality. We assessed clinical presentations, autoantibody specificities and therapeutic interventions in Native American (NA) patients with SLE. Methods: Patients with SLE meeting 1997 American College of Rheumatology classification criteria (n=3148) were enrolled between 1992 and 2010 in the multiethnic, cross-sectional Lupus Family Registry and Repository. Clinical, demographic and therapeutic information were extracted from medical records using a standardised form and formalised training. Autoantibodies were assessed by indirect immunofluorescence (antinuclear antibodies (ANA) and antidouble-stranded DNA), precipitin (ENA) and ELISA (IgG and IgM anticardiolipins). Results: NA patients met SLE classification at a younger age (29.89±12.3 years) than European Americans (EA; 32.02±12.87, P=0.0157) and a similar age to African-Americans (AAs) and Hispanics (HIS). More NA patients had concurrent rheumatic diseases or symptoms, such as Raynaud's phenomenon, interstitial lung disease, Sjögren's syndrome and systemic sclerosis. Compared with EAs, NAs were more likely to have high-titre ANA (≥1:3240; P

Objectives Epstein-Barr virus (EBV) is considered an important environmental factor in SLE aetiology, but the relationship between SLE and EBV in the Filipino population is unknown. We tested associations between SLE, lupus-associated autoantibodies and seropositivity for EBV and other herpes viruses in the Filipino population. Methods Sera from Filipino patients with SLE (n=233), unaffected first-degree relatives (FDRs, n=543) and unrelated controls (n=221) were tested for antibodies against EBV, cytomegalovirus (CMV) and herpes simplex viruses (HSV-1 and HSV-2) by standardised ELISAs. Humoral specificity against EBV nuclear antigen (EBNA)-1 was compared by solid-phase epitope mapping. Autoantibodies were detected by a bead-based multiplex assay. Results were analysed by Fisher's exact test, Student's t-test, χ2 test and one-way analysis of variance, as appropriate for the question. Results Filipino patients with SLE had increased seroprevalence and elevated antibody concentrations against EBV viral capsid antigen (EBV-VCA), CMV, HSV-1 and HSV-2 compared with unrelated controls (p

Systemic lupus erythematosus (SLE) etiology likely involves complex interactions between genetic and environmental stimuli that lead to autoantibody production and chronic inflammation. Infections are considered likely environmental triggers of SLE because certain viruses and bacteria can stimulate the innate immune system, incite the production of autoreactive responses, and alter immune cell activation and regulation. Specifically, decades of research point to Epstein–Barr virus (EBV) contributing to lupus pathogenesis. EBV promotes the development of lupus-associated autoantibodies through molecular mimicry and epitope spreading, and EBV proteins can drive aberrant activation of B cells and cytotoxic T cells by mimicking the functional effects of CD40 and interleukin-10. In individuals who are inherently susceptible to immune dysregulation, such microbe-driven changes can enhance the onset or progression of autoimmune disease. Microbiota may also influence autoimmunity. Finally, some infections may protect against autoimmunity in animal models, highlighting the intricacy of the gene–environment interactions that influence SLE pathogenesis.

Friedreich ataxia is caused by an expanded (GAA•TTC)n sequence, which is unstable during intergenerational transmission and in most patient tissues, where it frequently undergoes large deletions. We investigated the effect of DSB repair on instability of the (GAA•TTC)n sequence. Linear plasmids were transformed into Escherichia coli so that each colony represented an individual DSB repair event. Repair of a DSB within the repeat resulted in a dramatic increase in deletions compared with circular templates, but DSB repair outside the repeat tract did not affect instability. Repair-mediated deletions were independent of the orientation and length of the repeat, the location of the break within the repeat or the RecA status of the strain. Repair at the center of the repeat resulted in deletion of approximately half of the repeat tract, and repair at an off-center location produced deletions that were equivalent in length to the shorter of the two repeats flanking the DSB. This is consistent with a single-strand annealing mechanism of DSB repair, and implicates erroneous DSB repair as a mechanism for genetic instability of the (GAA•TTC)n sequence. Our data contrast significantly with DSB repair within (CTG•CAG)n repeats, indicating that repair-mediated instability is dependent on the sequence of the triplet repeat.

Systemic lupus erythematosus (SLE) is often preceded by immune dysregulation and clinical manifestations below the threshold for SLE classification. This review discusses current and evolving concepts about the preclassification period of SLE, including clinical and mechanistic observations, and potential avenues for early identification and intervention.Although incomplete lupus erythematosus (ILE) involves fewer clinical manifestations than SLE, ILE patients can suffer organ damage and early mortality. Common clinical features in ILE include antinuclear antibody seropositivity, polyarthritis, immunologic manifestations, and hematological disorders. Despite having lower disease activity and damage scores than SLE patients, ILE patients may develop pulmonary arterial hypertension, or renal, neurological, or peripheral vascular damage. The recently proposed Systemic Lupus International Collaborating Clinics SLE classification criteria could shift the period considered 'preclinical SLE'. Murine studies suggest that the balance of T-helper/T-regulatory cells, peroxisome proliferator-activated receptor γ activity, and plasmacytoid dendritic cell pathways may be valuable targets for early intervention.Advances in our understanding of early SLE, including stages before clinical features are fully developed, will improve our ability to identify individuals at high risk of classification for potential prevention trials, provide necessary information to improve diagnostic testing, and perhaps identify novel targets for directed therapeutics in clinical SLE.

Objective SLE is traditionally classified using the American College of Rheumatology (ACR) criteria. The Systemic Lupus International Collaborating Clinics (SLICC) recently validated an alternative system. This study examined large cohorts of subjects with SLE and incomplete lupus erythematosus (ILE) to compare the impact of ACR and SLICC criteria. Methods Medical records of subjects in the Lupus Family Registry and Repository were reviewed for documentation of 1997 ACR classification criteria, SLICC classification criteria and medication usage. Autoantibodies were assessed by indirect immunofluorescence (ANA, antidouble-stranded DNA), precipitin (Sm) and ELISA (anticardiolipin). Other relevant autoantibodies were detected by precipitin and with a bead-based multiplex assay. Results Of 3575 subjects classified with SLE under at least one system, 3312 (92.6%) were classified as SLE by both systems (SLE both ), 85 only by ACR criteria (SLE ACR-only ) and 178 only by SLICC criteria (SLE SLICC-only ). Of 440 subjects meeting 3 ACR criteria, 33.9% (149/440) were SLE SLICC-only , while 66.1% (n=291, designated ILE) did not meet the SLICC classification criteria. Under the SLICC system, the complement criterion and the individual autoantibody criteria enabled SLE classification of SLE SLICC-only subjects, while SLE ACR-only subjects failed to meet SLICC classification due to the combined acute/subacute cutaneous criterion. The SLICC criteria classified more African-American subjects by the leucopenia/lymphopenia criterion than did ACR criteria. Compared with SLE ACR-only subjects, SLE SLICC-only subjects exhibited similar numbers of affected organ systems, rates of major organ system involvement (∼30%: pulmonary, cardiovascular, renal, neurological) and medication history. Conclusions The SLICC criteria classify more subjects with SLE than ACR criteria; however, individuals with incomplete lupus still exist under SLICC criteria. Subjects who gain SLE classification through SLICC criteria exhibit heterogeneous disease, including potential major organ involvement. These results provide supportive evidence that SLICC criteria may be more inclusive of SLE subjects for clinical studies.

Copyright @ 2012 Bourn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)(n) sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)(n) sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)(n) sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)(n) sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)(n) sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway. IDB was supported by a postdoctoral fellowship from the National Ataxia Foundation. RMP was supported by Ataxia UK. SA was supported by The Wellcome Trust. This research was made possible by grants from the National Institutes of Health (NIH/NINDS) and the Muscular Dystrophy Association to S.I.B.

Expanded triplet repeat sequences are known to cause at least 16 inherited neuromuscular diseases. In addition to short-length changes, expanded triplet repeat tracts frequently undergo large changes, often amounting to hundreds of base-pairs. Such changes might occur when template or primer slipping creates insertion/deletion loops (IDLs), which are normally repaired by the mismatch repair system (MMR). However, in prokaryotes and eukaryotes, MMR promotes large changes in the length of (CTG·CAG) n sequences, the motif most commonly associated with human disease. We tested the effect of MMR on instability of the expanded (GAA·TTC) n sequence, which causes Friedreich ataxia, by comparing repeat instability in wild-type and MMR-deficient strains of Escherichia coli . As expected, the prevalence of small mutations increased in the MMR-deficient strains. However, the prevalence of large contractions increased in the MMR mutants specifically when GAA was the lagging strand template, the orientation in which replication fork stalling is known to occur. After hydroxyurea-induced stalling, both orientations of replication showed significantly more large contractions in MMR mutants than in the wild-type, suggesting that fork stalling may be responsible for the large contractions. Deficiency of MMR promoted large contractions independently of RecA status, a known determinant of (GAA·TTC) n instability. These data suggest that two independent mechanisms act in response to replication stalling to prevent instability of the (GAA·TTC) n sequence in E. coli , when GAA serves as the lagging strand template: one that is dependent on RecA-mediated restart of stalled forks, and another that is dependent on MMR-mediated repair of IDLs. While MMR destabilizes the (CTG·CAG) n sequence, it is involved in stabilization of the (GAA·TTC) n sequence. The role of MMR in triplet repeat instability therefore depends on the repeat sequence and the orientation of replication.