Objective. New examples support the concept that host immune responses to pathogenic organisms can act as the nidus for autoimmunity. Two such examples implicate the Epstein-Barr virus (EBV) in systemic lupus erythematosus (SLE), i.e., data consistent with SLE anti-Sm and anti-60-kd Ro autoantibodies emerging from distinct humoral immune responses to Epstein-Barr nuclear antigen 1 (EBNA-1). We undertook this study to further test whether the humoral immune response to EBNA-1 is a risk factor for pediatric SLE.Methods. Sera from pediatric lupus patients and healthy matched controls were tested for anti-EBNA-1 by Western blotting and enzyme-linked immunosorbent assay (ELISA). To define the fine specificity of their anti-EBNA-1 humoral immune response, fragments of EBNA-1 and the maximally overlapping unique octapeptides of EBNA-1 were tested by modified ELISAs.Results. All 36 pediatric SLE patient sera tested recognized EBNA-1, while sera from only 25 of 36 matched EBV-positive controls targeted EBNA-1 (P < 0.005). Epitope mapping revealed that the humoral anti-EBNA-1 response in pediatric SLE was distinct from and less restricted than that in matched normal individuals. Meanwhile, no significant differences between SLE patient sera and control sera were observed in the responses to other herpesviruses or in binding to sequential epitopes from cytomegalovirus immediateearly antigen or EBNA-2.Conclusion. Anti-EBNA-1 antibodies are associated with pediatric-onset SLE. Furthermore, an altered humoral immune response to EBNA-1, characteristic of SLE, has been found and may be an important SLE susceptibility factor.Systemic lupus erythematosus (SLE) is a chronic, systemic, idiopathic, clinically heterogeneous autoimmune disease. Nearly all SLE patients have high titers of serum autoantibodies (1,2) that usually precede the onset of clinical SLE by many years (3). Many potential environmental factors in disease onset have been evaluated, although consensus supporting a particular unifying susceptibility factor has not been obtained (4).Epstein-Barr virus (EBV) infection is one of the environmental risk factors most closely associated with SLE. Virtually all pediatric SLE patients have serologic evidence of EBV infection, while only 70% of pediatric controls are seropositive (odds ratio [OR] 49.9, 95% confidence interval [95% CI] 9.3-1,025, P Ͻ 0.0001), a result that is confirmed at the level of recovered EBV DNA (5,6) and in antinuclear antibody (ANA)-positive adult SLE patients (7).EBV appears well suited to initiate autoimmune disease. EBV infects B cells and promotes dysregulated polyclonal B cell activation and autoantibody production (8-11). EBV latency provides lifelong antigenic chal-

Objective The replacement of standard immunofluorescence anti-nuclear antibody (ANA) methods with bead-based assays is a new clinical option. A large, multi-racial cohort of SLE patients, blood relatives and unaffected control individuals was evaluated for familial aggregation and subset clustering of autoantibodies by high-throughput serum screening technology and traditional methods. Methods Serum samples (1,540 SLE patients, 1,127 unaffected relatives, and 906 healthy, population-based controls) were analyzed for SLE autoantibodies using a bead-based assay, immunofluorescence, and immunodiffusion. Autoantibody prevalence, disease sensitivity, clustering, and association with standard immunodiffusion results were evaluated. Results ANA frequency in SLE patient sera were 89%, 73%, and 67% by BioPlex 2200 and 94%, 84%, and 86% by immunofluorescence in African-American, Hispanic, and European-American patients respectively. 60kD Ro, La, Sm, nRNP A, and ribosomal P prevalence were compared across assays, with sensitivities ranging from 0.92 to 0.83 and specificities ranging from 0.90 to 0.79. Cluster autoantibody analysis showed association of three subsets: 1) 60kD Ro, 52kD Ro and La, 2) spliceosomal proteins, and 3) dsDNA, chromatin, and ribosomal P. Familial aggregation of Sm/RNP, ribosomal P, and 60kD Ro in SLE patient sibling pairs was observed (p ≤ 0.004). Simplex pedigree patients had a greater prevalence for dsDNA (p=0.0003) and chromatin (p=0.005) autoantibodies than multiplex patients. Conclusion ANA frequencies detected by a bead-based assay are lower in European-American SLE patients compared to immunofluorescence. These assays have strong positive predictive values across racial groups, provide useful information for clinical care, and provide unique insights to familial aggregation and autoantibody clustering.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, humoral autoimmune disorder. The unifying feature among SLE patients is the production of large quantities of autoantibodies. Serum samples from 129 patients collected before the onset of SLE and while in the United States military were evaluated for early pre-clinical serologic events. The first available positive serum sample frequently already contained multiple autoantibody specificities (65%). However, in 34 SLE patients the earliest pre-clinical serum sample positive for any detectable common autoantibody bound only a single autoantigen, most commonly 60 kD Ro (29%), nRNP A (24%), anti-phospholipids (18%) or rheumatoid factor (15%). We identified several recurrent patterns of autoantibody onset using these pre-diagnostic samples. In the serum samples available, anti-nRNP A appeared before or simultaneously with anti-nRNP 70 K in 96% of the patients who had both autoantibodies at diagnosis. Anti-60 kD Ro antibodies appeared before or simultaneously with anti-La (98%) or anti-52 kD Ro (95%). The autoantibody response in SLE patients begins simply, often binding a single specific autoantigen years before disease onset, followed by epitope spreading to additional autoantigenic specificities that are accrued in recurring patterns.

Background Prolidase deficiency is a rare autosomal recessive disease in which one of the last steps of collagen metabolism, cleavage of proline-containing dipeptides, is impaired. Only about 93 patients have been reported with about 10% also having systemic lupus erythematosus (SLE). Methods We studied a large extended Amish pedigree with four prolidase deficiency patients and three heterozygous individuals for lupus-associated autoimmunity. Eight unaffected Amish children served as normal controls. Prolidase genetics and enzyme activity were confirmed. Antinuclear antibodies (ANA) were determined using indirect immunofluorescence and antibodies against extractable nuclear antigens were determined by various methods, including double immunodiffusion, immunoprecipitation and multiplex bead assay. Serum C1q levels were determined by enzyme-linked immunosorbent assay. Results Two of the four homozygous prolidase deficiency subjects had a positive ANA. One had anti-double-stranded DNA, while another had precipitating anti-Ro. By the simultaneous microbead assay, three of the four had anti-Sm and anti-chromatin. One of the three heterozygous subjects had a positive ANA and immunoprecipitation of a 75 000 molecular weight protein. The unaffected controls had normal prolidase activity and were negative for autoantibodies. Conclusions Prolidase deficiency may be associated with the loss of immune tolerance to lupus-associated autoantigens even without clinical SLE.

Purpose. This study evaluates high-throughput autoantibody screening and determines associated systemic lupus erythematosus (SLE) clinical features in a large lupus cohort. Methods. Clinical and demographic information, along with serum samples, were obtained from each SLE study participant after appropriate informed consent. Serum samples were screened for 10 distinct SLE autoantibody specificities and examined for association with SLE ACR criteria and subcriteria using conditional logistic regression analysis. Results. In European-American SLE patients, autoantibodies against 52 kD Ro and RNP 68 are independently enriched in patients with lymphopenia, anti-La, and anti-ribosomal P are increased in patients with malar rash, and anti-dsDNA and anti-Sm are enriched in patients with proteinuria. In African-American SLE patients, cellular casts associate with autoantibodies against dsDNA, Sm, and Sm/nRNP. Conclusion. Using a high-throughput, bead-based method of autoantibody detection, anti-dsDNA is significantly enriched in patienets with SLE ACR renal criteria as has been previously described. However, lymphopenia is associated with several distinct autoantibody specificities. These findings offer meaningful information to allow clinicians and clinical investigators to understand which autoantibodies correlate with select SLE clinical manifestations across common racial groups using this novel methodology which is expanding in clinical use.