Antibodies to histone in the pediatric population: a retrospective chart review.

BACKGROUND: Antibodies to histone have been associated in the adult literature with systemic lupus erythematosus(SLE) and drug induced lupus(DILE). Little data is available regarding the spectrum of pathology that antibodies to histone encompass in the pediatric population. Prior studies suggest an association with SLE, juvenile idiopathic arthritis(JIA), uveitis and linear scleroderma. METHODS: Patient charts were reviewed that contained positive anti-histone antibody testing during a consecutive three year period. Patient diagnosis along with the presence of: anti-histone antibody titer, ANA, and the presence of other autoantibodies to SSA, SSB, Sm, RNP, dsDNA and chromatin were obtained. The frequency of SLE, JIA and DILE was further investigated in specific subsets. RESULTS: 139 individual charts were reviewed containing 41 different diagnoses. The most common diagnosis was hypermobility arthralgia with 22 patients. The most frequent rheumatologic diagnosis was JIA(nonsystemic) with 19. 13 patients in this study were diagnosed with SLE and 2 with DILE. 18 patients had other autoantibody production, of these, 11 had SLE or DILE. Only one of 62 patients with a weak antihistone antibody titer(1.0-1.5) was diagnosed with SLE. When strong titers are present(> 2.5), the antihistone antibody test was associated with a greater than 50% incidence of an underlying rheumatologic disease and ten times higher incidence of SLE than a weak titer. In regards to the frequency of SLE, there was a statistically significant difference between weak and moderate titers and between weak and strong titers. CONCLUSION: The presence of anti-histone antibody was observed in a variety of diagnoses in the pediatric population. Overall, the presence of anti-histone antibodies appears to have poor diagnostic utility for any specific condition. However, diagnostic utility for SLE does appear to improve with higher titers, when combined with other autoantibody positivity. Strength of titer did not appear to be a factor for JIA, but was the most frequently observed rheumatologic disease in this study.

Laboratory Studies in Autoimmune Diseases.

Autoimmune diseases (AID) are a great percentage of the patients needing to be seen by Rheumatologists. Their initial work-up by their primary care physician (PCP) can be helpful in determining who needs to be seen in consultation. If interpreted properly, initial laboratory studies can help the PCP know when to initiate a consultation. The following laboratory studies as outlined will provide the PCP with the necessary information to determine if a further AID work-up is indicated.

Azathioprine therapy for steroid-resistant Henoch-Schönlein purpura: a report of 6 cases.

BACKGROUND: A small percentage of children with Henoch-Schönlein purpura (HSP) develop a chronic form of the disease that often requires prolonged corticosteroid therapy. Disease modifying anti-rheumatic agents (DMARDs) or biologics have been successfully used to treat those refractory cases. Azathioprine is a DMARD that has been reported to be effective in HSP nephritis and in adult cutaneous leukocytoclastic vasculitis, a condition with cutaneous histology similar to HSP. CASE PRESENTATION: A description of 6 cases with relapsing HSP without significant renal involvement, treated with azathioprine are reported. All 6 cases met the classification criteria for the diagnosis of HSP, had relapsing symptoms despite corticosteroid use, were successfully treated with azathioprine and were tapered off of corticosteroids. The duration of azathioprine therapy ranged from 7-21 months and no adverse events were reported. CONCLUSIONS: Azathioprine is effective in controlling prolonged relapsing symptoms of HSP, allowing earlier discontinuation of corticosteroids. This report shows that azathioprine can be included in the therapeutic options for relapsing HSP and is the first case series in the literature of azathioprine use in HSP without significant renal involvement.

Immune Complexes in Juvenile Idiopathic Arthritis.

Juvenile idiopathic arthritis (JIA) reflects a group of clinically heterogeneous, autoimmune disorders in children characterized by chronic arthritis and hallmarked by elevated levels of circulating immune complexes (CICs) and associated complement activation by-products in their sera. Immune complexes (ICs) have been detected in patients' sera with JIA utilizing a variety of methods, including the anti-human IgM affinity column, C1q solid-phase assay, polyethylene glycol precipitation, Staphylococcal Protein A separation method, anti-C1q/C3 affinity columns, and FcγRIII affinity method. As many as 75% of JIA patients have had IC detected in their sera. The CIC proteome in JIA patients has been examined to elucidate disease-associated proteins that are expressed in active disease. Evaluation of these ICs has shown the presence of multiple peptide fragments by SDS-PAGE and 2-DE. Subsequently, all isotypes of rheumatoid factor (RF), isotypes of anti-cyclic citrullinated peptide (CCP) antibodies, IgG, C1q, C4, C3, and the membrane attack complex (MAC) were detected in these IC. Complement activation and levels of IC correlate with disease activity in JIA, indicating their role in the pathophysiology of the disease. This review will summarize the existing literature and discuss the role of possible protein modification that participates in the generation of the immune response. We will address the possible role of these events in the development of ectopic germinal centers that become the secondary site of plasma cell development in JIA. We will further address possible therapeutic modalities that could be instituted as a result of the information gathered by the presence of ICs in JIA.

FcγRIIIa-Syk Co-signal Modulates CD4+ T-cell Response and Up-regulates Toll-like Receptor (TLR) Expression.

CD4(+) T-cells in systemic lupus erythematosus (SLE) patients show altered T-cell receptor signaling, which utilizes Fc-receptor γ-chain FcRγ-Syk. A role for FcγRIIIa activation from immune complex (IC) ligation and sublytic terminal complement complex (C5b-9) in CD4(+) T-cell responses is not investigated. In this study, we show that the ICs present in SLE patients by ligating to FcγRIIIa on CD4(+) T-cells phosphorylate Syk and provide a co-stimulatory signal to CD4(+) T-cells in the absence of CD28 signal. This led to the development of pathogenic IL-17A(+) and IFN-γ(high) CD4(+) T-cells in vitro. Cytokines IL-1ß, IL-6, TGF-ß1, and IL-23 were the only requirement for the development of both populations. SLE patients CD4(+) T-cells that expressed CD25, CD69, and CD98 bound to ICs showed pSyk and produced IFN-γ and IL-17A. This FcγRIIIa-mediated co-signal differentially up-regulated the expression of IFN pathway genes compared with CD28 co-signal. FcγRIIIa-pSyk up-regulated several toll-like receptor genes as well as the HMGB1 and MyD88 gene transcripts. ICs co-localized with these toll-like receptor pathway proteins. These results suggest a role for the FcγRIIIa-pSyk signal in modulating adaptive immune responses.

Laboratory Evaluation in Pediatric Autoimmune Diseases.

Pediatric autoimmune diseases are chronic lifelong disorders associated with potential disability and increased morbidity and mortality if not properly recognized and treated. On the basis of largely expert opinion in addition to observational studies, children with suspected autoimmune disease should undergo general laboratory and autoantibody screening. (1)(2)(4)(6)(7)(11) There can be many causes of positive antinuclear antibody test results, including, but not limited to, autoimmune disease. On the basis of expert opinion and observational studies, a thorough history and physical examination as well as laboratory evaluation is recommended (often in consultation with a pediatric rheumatologist) to elucidate the cause for a positive test result. (4) (6)(11)

Induced expression of FcγRIIIa (CD16a) on CD4+ T cells triggers generation of IFN-γhigh subset.

Whether or not CD4(+) T-cells express low affinity receptor FcγRIIIa (CD16a) in disease pathology has not been examined in great detail. In this study, we show that a subset of activated CD4(+) T-cells in humans express FcγRIIIa. The ligation of FcγRIIIa by immune complexes (ICs) in human CD4(+) T-cells produced co-stimulatory signal like CD28 that triggered IFN-γ production. The induced expression of FcγRIIIa on CD4(+) helper T-cells is an important finding since these receptors via ITAM contribute to intracellular signaling. The induced expression of FcγRIIIa on CD4(+) T helper cells and their ability to co-stimulate T-cell activation are important and novel findings that may reveal new pathways to regulate adaptive immune responses during inflammation and in autoimmunity.

Measurement and evaluation of isotypes of anti-citrullinated fibrinogen and anti-citrullinated alpha-enolase antibodies in juvenile idiopathic arthritis.

OBJECTIVES: Our objective was to evaluate sera from juvenile idiopathic arthritis (JIA) patients to investigate the presence of isotypes (IgA, IgG, IgM) of anti-citrullinated fibrinogen and anti-α-enolase antibodies and their association with rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody isotypes. METHODS: Sera were obtained from 89 JIA patients and were measured for isotypes (IgA, IgM) of anti-citrullinated and native fibrinogen and anti-α-enolase antibodies by enzyme-linked immunosorbent assay. Results were compared to anti-CCP antibody isotypes and RF isotypes, in addition to previously measured IgG anti-citrullinated fibrinogen and α-enolase antibodies. RESULTS: IgA anti-citrullinated fibrinogen antibodies were positive in 20 JIA patients and IgM in 11 JIA patients. Two IgM RF-positive polyarthritis patients were positive for all 3 isotypes of anti-citrullinated fibrinogen antibodies. IgA anti-citrullinated α-enolase antibodies were positive in 7 JIA patients and IgM in 9 JIA patients. IgA and IgG anti-citrullinated fibrinogen antibodies were commonly found in JIA patients positive for IgG anti-CCP antibodies and IgM RF. IgG anti-CCP antibodies and IgM RF levels were significantly higher in JIA patients with 3 or more anti-citrullinated autoantibody isotypes present. CONCLUSIONS: We have shown that isotypes of anti-citrullinated fibrinogen and α-enolase can be found in the serum of children with JIA of all onset types. Citrullinated autoantibody isotype diversity may indicate a more severe disease course in JIA patients.

Evaluation of anti-citrullinated type II collagen and anti-citrullinated vimentin antibodies in patients with juvenile idiopathic arthritis.

BACKGROUND: To determine the prevalence and significance of anti-citrullinated vimentin and anti-citrullinated type II collagen antibodies and elucidate their role in the disease process of juvenile idiopathic arthritis (JIA). METHODS: Sera were obtained from 95 patients with various subtypes of JIA, 19 systemic lupus erythematosus (SLE) patients, and 10 healthy children. Antibodies were measured in the sera against citrullinated and native type II collagen and vimentin (vim1-16 and vim 59-74) by enzyme-linked immunosorbent assay. Samples were compared to anti-cyclic citrullinated peptide (anti-CCP) antibody and rheumatoid factor (RF) isotypes, and our previously measured anti-citrullinated fibrinogen and α-enolase antibodies on the same patient population, in addition to erythrocyte sedimentation rate and C-reactive protein. The relationship between the anti-citrullinated antibody profile and disease activity and joint damage were also investigated. RESULTS: Twenty-three JIA patients (24%) demonstrated reactivity to anti-citrullinated type II collagen. Ten JIA patients (10.5%) demonstrated reactivity to anti-citrullinated vimentin 1-16 antibodies and 7 (7.4%) to anti-citrullinated vimentin 59-74 antibodies. One IgM RF-positive polyarticular patient was positive for all 5 of the citrullinated autoantibodies tested. Thirty-seven different subsets of patients were identified based on their anti-citrullinated autoantibody and RF isotype profile. No significant associations were noted with anti-citrullinated type II collagen and anti-citrullinated vimentin antibodies with joint damage or disease activity. Anti-citrullinated vimentin 59-74 antibodies demonstrated the highest overall specificity at 89.7%, with anti-citrullinated vimentin 1-16 and anti-citrullinated type II collagen antibodies at 86.2%. CONCLUSION: This study demonstrates that antibodies to multiple citrullinated epitopes are present in the sera of patients with various subtypes of JIA. It also demonstrates the frequent occurrence of anti-citrullinated type II collagen and anti-citrullinated fibrinogen antibodies. The presence of autoantibodies to citrullinated antigens in JIA patients is highly diverse.

Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints. METHODS: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography. RESULTS: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p<0.005). CONCLUSION: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.

The role of anti-cyclic citrullinated peptide (CCP) antibodies in early detection of rheumatoid arthritis: an overview of the INOVA Diagnostics, Inc. QUANTA Lite CCP assays.

INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and the presence of self-reactive autoantibodies. Since the discovery of anti-cyclic citrullinated peptide (anti-CCP) antibodies, several assays have been developed to measure these autoantibodies in RA patients. The first-generation kit offered high specificity, but sensitivity was low. The second-generation IgG anti-CCP antibody assay (CCP2) offered the same high specificity, with greatly improved sensitivity for RA. INOVA Diagnostics, Inc. offers, in addition to CCP2, a third-generation assay with higher sensitivity compared with CCP2 and also a combined IgG/IgA anti-CCP antibody assay. AREAS COVERED: The review covers the use of INOVA Diagnostics, Inc. multiple anti-CCP antibody assays in early detection of RA, while also comparing these assays with other commercially available methods of measuring anti-CCP antibodies. While most of the review focuses on the significance of these autoantibodies in adult RA patients, their role in juvenile idiopathic arthritis is also discussed. EXPERT OPINION: Detection of anti-CCP antibodies has emerged as one of the most important disease markers in RA patients. Several methods are available to measure anti-CCP antibodies, and isotyping and identification of citrullination targets are now the next step in further characterizing these autoantibodies.

Measurement of autoantibodies in pediatric-onset systemic lupus erythematosus and their relationship with disease-associated manifestations.

OBJECTIVE: To evaluate an autoantibody profile in pediatric-onset systemic lupus erythematosus (SLE) patients to determine clinical and statistical associations with disease-associated manifestations. METHODS: Sera from 53 SLE patients and 22 healthy individuals were collected. Antibodies to C1q, histone, chromatin, ribosomal P, dsDNA, and high-avidity dsDNA were measured by enzyme-linked immunosorbent assays. Patient records were evaluated for clinical and laboratory associations. RESULTS: The most prevalent autoantibodies found in the SLE cohort were anti-C1q antibodies (n = 32, 60%), which correlated significantly with proteinuria and decreased complement levels (P < 0.05). Anti-C1q and antihistone antibodies were significantly elevated in patients with class III/IV nephritis compared with class I/II/V nephritis (P = 0.041). SLE patients with active nephritis at the time of sample collection demonstrated significantly elevated levels of anti-C1q antibodies compared with those without active nephritis, also exhibiting 100% sensitivity for active nephritis, proteinuria, and urinary casts. Antibodies to C1q, dsDNA, histone, and chromatin were significantly elevated in patients with active disease (P < 0.01). Chart-documented anti-dsDNA antibodies were positive in 28 SLE patients, INOVA anti-dsDNA antibodies in 25 patients, and high-avidity anti-dsDNA antibodies in 8 patients. Antihistone correlated significantly with leukopenia and hemolytic anemia (P < 0.05). CONCLUSIONS: This study indicates the importance of measuring anti-C1q antibodies in pediatric-onset SLE patients because elevated anti-C1q antibodies may be more indicative of renal disease activity, showing significant correlation with proteinuria, urinary casts, and active nephritis. Antibodies to C1q, histone, chromatin, and dsDNA exhibited the strongest association with clinical features, indicating the importance of measuring all of these antibodies in pediatric-onset SLE patients.

Significance of complement components C1q and C4 bound to circulating immune complexes in juvenile idiopathic arthritis: support for classical complement pathway activation.

OBJECTIVES: Immune complexes (ICs) from sera of juvenile idiopathic arthritis (JIA) patients show increased complement opsonisation; however, a definitive role for involvement of the classical or alternative pathway is not entirely clear. To delineate the role of these pathways, we measured activated complement products bound to circulating IC (CICs) in the sera of JIA patients. METHODS: Sera from 100 JIA patients and 22 healthy children were collected. C1q, C4, C3, C3d, and membrane attack complex (MAC) bound to CICs were measured by enzyme-linked immunosorbent assay. Data was compared to IgM rheumatoid factor (RF), IgG anti-cyclic citrullinated peptide (CCP) antibodies, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) levels. RESULTS: Mean levels of C1q, C4, and MAC bound to CICs were significantly elevated in JIA patients compared to healthy children. C1q correlated significantly with C4 and MAC bound to CICs and C4 and MAC also demonstrated significant correlation. No significant differences were noted in complement components bound to CICs when evaluating IgM RF, anti-CCP antibody, and CRP positivity. A significant correlation was noted between MAC bound to CICs and ESR. C1q and MAC bound to CICs mean levels were significantly higher in patients with an elevated ESR compared to those with a normal ESR level. CONCLUSIONS: JIA patients have elevated levels of complement components bound to CICs, particularly from the classical pathway. Moreover, classical pathway components were associated with ESR, a marker of disease activity. MAC bound to CICs also correlated significantly with ESR, further supporting the notion of complement-mediated tissue injury that is triggered by IC-mediated classical pathway activation.

T cell activation by terminal complex of complement and immune complexes.

T cell hyperactivation and complement consumption are prominent features of the immunopathology of systemic lupus erythematosus. Although complement activation is secondary to autoantibodies that form immune complexes (ICs), the trigger for alterations in human peripheral blood T cells is poorly understood. To study the impact (on T cells) of several types of preformed ICs and terminal complement complex, also referred to as C5b-9, we incubated these immune reactants with peripheral blood naive CD4(+) T cells as well as Jurkat cells and analyzed their effects on cellular behavior. We first assembled the C5b-9 in situ on the membrane and observed its assembly primarily on a single site where it promoted aggregation of membrane rafts and recruitment of the CD3 signaling complex. However, C5b-9 alone did not initiate proliferation or commencement of downstream signaling events associated with T cell activation. When T cells were treated with ICs together with nonlytic C5b-9, changes associated with T cell activation by possible antigen engagement then occurred. T cell antigen receptor signaling proteins, including ζ-chain, ZAP-70, Syk, Src, and Lck, were phosphorylated and organized in a synapse-like structure. The cytoskeleton formed F-actin spindles and a distal pole complex, resulting in a bipolar distribution of phosphorylated ezrin-radixin-moesin and F-actin. Furthermore, ICs and nonlytic C5b-9 induced T cell proliferation and IFN-γ production. These results raise the possibility that ICs and the nonlytic C5b-9 modulate T cell-mediated responses in systemic lupus erythematosus and other related chronic inflammatory disorders.

Evidence of fibrinogen as a target of citrullination in IgM rheumatoid factor-positive polyarticular juvenile idiopathic arthritis.

BACKGROUND: Several studies have noted the significance of measuring anti-cyclic citrullinated peptide (CCP) antibodies in juvenile idiopathic arthritis (JIA) as an important indicator for destructive disease, as is the case in rheumatoid arthritis (RA). While the role of anti-CCP antibodies in RA and JIA has become better understood, the identity of the target proteins of this modification has remained elusive. In this study, we evaluated serum from patients with various subtypes of JIA to investigate the presence of anti-deiminated (citrullinated) fibrinogen and anti-citrullinated α-enolase antibodies, and their association with RF and anti-CCP antibody isotypes. METHODS: Sera were obtained from 96 JIA patients, 19 systemic lupus erythematosus (SLE) patients, and 10 healthy children. All sera were measured for antibodies against citrullinated and native fibrinogen and α-enolase by an enzyme linked immunosorbent assay (ELISA). In addition, all sera were assayed for anti-CCP antibody isotypes and rheumatoid factor (RF) isotypes by ELISA. The relationship between anti-citrullinated fibrinogen and anti-α-enolase antibodies and disease activity and joint damage were also investigated. All results were correlated with clinical and laboratory parameters using Spearman's rho correlation coefficient. Multiple logistic regression analysis was utilized to identify which variables were associated with joint erosions and diagnosis of JIA. RESULTS: Thirty-one JIA patients (32%) demonstrated reactivity to citrullinated fibrinogen and 9 (9%) to citrullinated α-enolase. Reactivity to citrullinated fibrinogen and α-enolase was predominantly found in IgM RF-positive polyarthritis patients. Fourteen JIA patients reacted with native α-enolase and a higher percentage of SLE patients reacted with citrullinated α-enolase when compared to JIA patients. Anti-citrullinated fibrinogen antibodies correlated with the presence of IgG anti-CCP antibodies and IgA and IgM RF. The presence of anti-citrullinated α-enolase antibodies correlated with IgA anti-CCP antibodies. IgG anti-CCP antibodies were significantly associated with joint damage and anti-citrullinated fibrinogen antibodies were strongly associated with JIA when compared to control groups. Anti-citrullinated fibrinogen antibodies demonstrated high sensitivity (81%) for IgM RF-positive polyarticular JIA. IgG anti-CCP antibodies had the highest specificity (95%) for JIA, with anti-citrullinated fibrinogen antibodies, IgA anti-CCP antibodies and IgA RF all following at 84%. CONCLUSIONS: JIA patient sera exhibited strong reactivity to anti-citrullinated fibrinogen antibodies and demonstrated high sensitivity and specificity for JIA, primarily in IgM RF-positive polyarthritis patients. Fibrinogen is one of several protein targets for citrullination in JIA.

Teriparatide [rhPTH (1-34)], but not strontium ranelate, demonstrated bone anabolic efficacy in mature, osteopenic, ovariectomized rats.

We compared teriparatide (TPTD) and strontium ranelate (SR) efficacy on bone formation activity in a mature rat model of estrogen-deficiency bone loss. Rats were ovariectomized (OVX) at age 6 months and permitted to lose bone for 2 months to establish osteopenia before initiation of treatment with TPTD (5 or 15 µg/kg · d sc) or SR (150 or 450 mg/kg · d oral gavage). After 3 wk, RT-PCR analyses of bone formation genes in the distal femur metaphysis showed significant elevation of collagen 1α2, osteocalcin, bone sialoprotein, alkaline phosphatase, and Runx2 gene expression at both TPTD doses, relative to OVX controls. SR had no significant effect on expression of these genes. TPTD treatment for 12 wk dose dependently increased lumbar vertebral (LV) and femoral midshaft bone mineral content (BMC) and bone mineral density over pretreatment and age-matched OVX controls. SR 150 increased BMC, and SR 450 increased BMC and bone mineral density of femoral midshaft and LV over OVX controls. There were significant dose-dependent TPTD increases of LV and femoral neck strength, and TPTD 15 also increased midshaft strength compared with pretreatment and age-matched OVX controls. SR did not enhance bone strength relative to pretreatment or age-matched OVX controls. Histomorphometry of the proximal tibial metaphysis showed dose-dependent effects of TPTD on trabecular area, number, width, and osteoblast surface, bone mineralizing surface, and bone formation rate relative to pretreatment and age-matched OVX controls, whereas SR had no effect on these parameters. These findings confirmed the bone anabolic efficacy of teriparatide, but not SR in mature, osteopenic, OVX rats.

Novel 3-aryl indoles as progesterone receptor antagonists for uterine fibroids.

We report the synthesis and characterization of novel 3-aryl indoles as potent and efficacious progesterone receptor (PR) antagonists with potential for the treatment of uterine fibroids. These compounds demonstrated excellent selectivity over other steroid nuclear hormone receptors such as the mineralocorticoid receptor (MR). They were prepared from 2-bromo-6-nitro indole in four to six steps using a Suzuki cross-coupling as the key step. Compound 8f was orally active in the complement 3 model of progesterone antagonism in the rat uterus and demonstrated partial antagonism in the McPhail model of progesterone activity.

Proteomic analysis of circulating immune complexes in juvenile idiopathic arthritis reveals disease-associated proteins.

Juvenile idiopathic arthritis reflects a group of clinically heterogeneous arthritides hallmarked by elevated concentrations of circulating immune complexes. In this study, the circulating immune complex proteome was examined to elucidate disease-associated proteins that are overexpressed in patients with an aggressive, and at times destructive, disease phenotype. To solve this proteome, circulating immune complexes were isolated from the sera of patients with chronic, erosive or early-onset, aggressive disease and from patients in medical remission or healthy controls subsequent to protein separation by 2-DE. Thirty-seven protein spots were overexpressed in the circulating immune complexes of the aggressive disease groups as compared to controls, 28 of which have been confidently identified to date. Proteolytic fragments of glyceraldehyde-3-phosphate dehydrogenase, serotransferrin, and α-1-antitrypsin have been identified among others. In total, these 28 putative disease-associated proteins most definitely contribute to immune complex formation and likely have a significant role in disease etiology and pathogenesis. Moreover, these proteins represent markers of aggressive disease, which could aid in diagnosis and management strategies, and potential therapeutic targets to prevent or control disease outcome. This is the first in-depth analysis of the circulating immune complex proteome in juvenile idiopathic arthritis.