An altered immune response to Epstein-Barr nuclear antigen 1 in pediatric systemic lupus erythematosus

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 immediate-early 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 in adults is associated with previous Epstein‐Barr virus exposure

Objective

The possible molecular mimicry of the Epstein‐Barr virus (EBV) peptide PPPGRRP by the peptide PPPGMRPP from Sm B′/B of the human spliceosome is consistent with the possibility that EBV infection is related to the origin of systemic lupus erythematosus (SLE) in some patients. Association of EBV exposure with SLE was therefore tested for and subsequently found in children and adolescents (odds ratio [OR] 49.9, 95% confidence interval [95% CI] 9.3–1,025, P < 10⁻¹¹). These results were confirmed at the level of EBV DNA (OR > 10, 95% CI 2.53–∞, P < 0.002). Much smaller seroconversion rate differences were found against 4 other herpes viruses. Herein, we extend these studies to adults and test the hypothesis that EBV infection is associated with adult SLE.

Methods

We selected 196 antinuclear antibody–positive adult SLE patients (age ≥20 years) and 2 age‐, race‐, and sex‐matched controls per patient. SLE patients and matched controls were tested for evidence of previous infection with EBV, cytomegalovirus (CMV), herpes simplex virus types 1 and 2 (HSV‐1 and HSV‐2), or varicella‐zoster virus (VZV) by standardized enzyme‐linked immunosorbent assays.

Results

Of the 196 lupus patients tested, all but 1 had been exposed to EBV, while 22 of the 392 controls did not have antibodies consistent with previous EBV exposure (OR 9.35, 95% CI 1.45–∞, P = 0.014). No differences were observed between SLE patients and controls in the seroconversion rate against CMV, HSV‐2, or VZV.

Conclusion

These new data from adults, along with the many suggestive features of EBV infection, are consistent with the contribution of this infection to the etiology of SLE.

     

Cell-mediated immune response in systemic lupus erythematosus.

Cell-mediated immunity in 29 patients with systemic lupus erythematosus was assayed by macrophage inhibitory test (MI-test) using native DNA as an antigen. The mean percentage migration in SLE and normal control was 77.8 and 98.1%. The percentage migration below 84.3% was considered to be a positive MI-test. Patients with SLE possessed cellular hypersensitivity to native DNA; positive MI-tests seemed to be present more often when the disease was clinically active. Percentage migration did not correlate with the presence of ANA and anti-DNA antibody. It is of note that lymphocytes from patients with negative ANA produced migration inhibitory factor in response to native DNA. The higher incidence of positive MI-tests in patients with nephrotic syndrome is impressive. The role of the presence of DNA-sensitized lymphocytes to the perpetuation of renal lesion in nephrotic syndrome is discussed.     

Association of Epstein‐Barr virus with systemic lupus erythematosus: Effect modification by race,age, and cytotoxic T lymphocyte–associated antigen 4 genotype

Objective

Epstein‐Barr virus (EBV) is hypothesized to play a role in the development of systemic lupus erythematosus (SLE). Cytotoxic T lymphocyte–associated antigen 4 (CTLA‐4) is important in regulating T cell–mediated immunity, encompassing the first line of response to viral infections, and genetic variation in CTLA‐4 has been associated with SLE. This study examined the seroprevalence of EBV in a population‐based study of SLE patients from the southeastern United States, and potential interactions with CTLA‐4 polymorphisms were assessed.

Methods

Cases comprised 230 subjects recently diagnosed as having SLE (144 African American and 86 white) from university and community‐based clinics, and controls comprised 276 age‐, sex‐, and state‐matched subjects (72 African American and 204 white) recruited from driver's license registries. Antibodies to EBV capsid antigen were determined by enzyme‐linked immunosorbent assay, with results expressed as positive or negative using the international standardized ratio (ISR) (a ratio of the sample absorbance to a known standard). CTLA‐4 genotypes were identified by polymerase chain reaction–based methods.

Results

In African Americans, EBV‐IgA seroprevalence was strongly associated with SLE (odds ratio [OR] 5.6, 95% confidence interval [95% CI] 3.0–10.6). In whites, the modest association of SLE with EBV‐IgA (OR 1.6) was modified by age, in that the strongest association was observed in those older than age 50 years (OR 4.1, 95% CI 1.6–10.4). The seroprevalence of EBV‐IgM and that of EBV‐IgG were not associated with SLE. Higher EBV‐IgG absorbance ratios were observed in SLE patients, with a significant dose response across units of the ISR in African Americans (P < 0.0001). Allelic variation in the CTLA‐4 gene promoter (−1661A/G) significantly modified the association between SLE and EBV‐IgA (P = 0.03), with a stronger association among those with the −1661AA genotype.

Conclusion

These findings suggest that repeated or reactivated EBV infection, which results in increased EBV‐IgA seroprevalence and higher IgG antibody titers, may be associated with SLE, and that the CTLA‐4 genotype influences immune responsiveness to EBV in SLE patients. The observed patterns of effect modification by race, age, and CTLA‐4 genotype should be examined in other studies and may help frame new hypotheses regarding the role of EBV in SLE etiology.

     

Survivorship in systemic lupus erythematosus. effect of antibody to extractable nuclear antigen

The course of 81 patients with systemic lupus erythematosus (SLE) who had sera tested for antibody to extractable nuclear antigen (ENA) was studied to determine the effect of the presence of antiENA antibody on survivorship. There were no differences in percent survival between the patients with and without antibody to ENA or those with and without antibody to the ribonucleoprotein (RNP) component of ENA. We conclude that there is no prognostic advantage to the presence of either antiENA or antiRNP antibody in patients with SLE.     

Dyslipoproteinemia in pediatric systemic lupus erythematosus

Patients with systemic lupus erythematosus are at increased risk for premature atherosclerosis. We examined one possible etiologic factor, dyslipoproteinemia, both before and after corticosteroid therapy. We identified 2 distinct patterns of dyslipoproteinemia. One is attributable to active disease; the other is attributable, in part, to corticosteroid therapy. The dyslipoproteinemia of active disease consists of depressed high density lipoprotein cholesterol and apoprotein A-I with elevated very low density lipoprotein cholesterol and triglyceride, while the dyslipoproteinemia after corticosteroid therapy consists of increased total cholesterol, very low density lipoprotein cholesterol, and triglyceride. The possible pathophysiologic mechanisms responsible for these patterns, as well as the possible roles in premature atherosclerosis seen in systemic lupus erythematosus patients, are discussed.     

Flares in pediatric systemic lupus erythematosus

OBJECTIVE: To determine the flare rate and the change in Safety of Estrogens in Lupus Erythematosus: National Assessment Systemic Lupus Erythematosus Disease Activity Index (SELENA SLEDAI) score with disease flare in pediatric systemic lupus erythematosus (pSLE). METHODS: A retrospective chart review of 62 patients with pSLE (ages 5-20 yrs). A flare was defined as the start of, or increase in, the dose of corticosteroids and/or the addition of an immunosuppressive medication. All pre-flare, flare, and post-flare visits were recorded with a SELENA SLEDAI score calculated for each visit. The flare rate was calculated by dividing the total number of flares in the cohort by the total followup years. RESULTS: Sixty-two patients were eligible. Forty-seven patients had 112 flares. The average number of flares/patient was 1.8 +/- 2.0 and the mean inter-flare time was 15.4 +/- 17.9 months. The flare rate in pSLE was 0.46 flares/patient-year of followup. The median time to first flare from the date of diagnosis was 14.3 months. Patients with cytopenia, pleuritis, or pericarditis, or a positive antibody to Smith nuclear antigen at the time of diagnosis had a significantly higher flare rate than those who did not. The average SELENA SLEDAI score at presentation was 12.5 +/- 5.4, at the pre-flare visit 6.3 +/- 3.5, and during a flare 7.9 +/- 5.1. CONCLUSION: This is the first large study to report a flare rate (0.46 flares/patient-year of followup) in pSLE. The flare rate was similar to what has been reported in pSLE previously but significantly lower than that reported in adults with lupus. The average change in the SELENA SLEDAI score with disease flare is 2 points.     

Refractory immune thrombocytopenia in systemic lupus erythematosus: response to mycophenolate mofetil

Immune thrombocytopenia (IT) is a common manifestation of systemic lupus erythematosus (SLE). Although severe IT (<20 x 10(9)/L) occurs in about 5-10% of patients, usually in the context of active disease, the absence of randomized controlled trials has not allowed the development of evidence-based guidelines for managing this condition. Conventionally, high-dose glucocorticoids are considered first-line therapy. Adjunctive medical and surgical treatments for patients with an absent or partial response to glucocorticoids have met with varying degrees of success. We describe an SLE patient with IT refractory to high-dose corticosteroids, pulse methylprednisolone and intravenous immunoglobulin therapy, whose platelet counts normalized during therapy with mycophenolate mofetil (MMF). Pending further controlled studies to confirm this observation, we suggest that MMF may be considered as a therapeutic option in the treatment of glucocorticoid-refractory immune thrombocytopenia in SLE.     

Altered immune response to glycine-rich sequences of Epstein-Barr nuclear antigen-1 in patients with rheumatoid arthritis and systemic lupus erythematosus

Prior studies have shown that patients with rheumatoid arthritis (RA) have an increased number of circulating Epstein-Barr virus-infected B lymphocytes and elevated titers of antibody to Epstein-Barr nuclear antigen-1 (EBNA-1), the major nuclear antigen expressed in latently infected B cells. However, it is not known whether antibodies from RA patients recognize the same epitopes as antibodies from normal subjects. are directed at the glycine-alanine repeating region of the molecule. Antibodies specific for this region are also somewhat more prevalent in RA patients than in normal subjects. A panel of synthetic peptides derived from EBNA-1 was used to analyze the immune response to antigenic epitopes outside the glycine-alanine region, using the peptides as solid-phase antigen. Sera from RA patients and from systemic lupus erythematosus patients contained elevated levels of IgG antibodies to 2 non-glycine-alanine peptide and to 3 non-glycine-alanine peptides, respectively. Two of the 3 peptides are glycine-rich, but antibodies that react with them are distinct from each other, as well as from those that react with the glycine-alanine epitope. Eight other peptides from the C-terminal portion of EBNA-1 either do not react with sera or show no difference between normal subjects and patient groups. The antibodies to the glycine-alanine peptide are enriched with kappa light chains, whereas antibodies to epitopes outside the glycine-alanine region are not so restricted among kappa and lambda light chains. Thus, RA patients and systemic lupus erythematosus patients have different antibody responses than do normal subjects, both quantitatively and qualitatively.     

Deiminated Epstein‐Barr virus nuclear antigen 1 is a target of anti–citrullinated protein antibodies in rheumatoid arthritis

Objective

To test the hypothesis that deimination of viral sequences containing Arg–Gly repeats could generate epitopes recognized by anti–citrullinated protein antibodies (ACPAs) that are present in rheumatoid arthritis (RA) sera.

Methods

Multiple antigen peptides derived from Epstein‐Barr virus (EBV)–encoded Epstein‐Barr nuclear antigen 1 (EBNA‐1) were synthesized, substituting the arginines with citrulline, and were used to screen RA sera. Anti–cyclic citrullinated peptide antibodies were purified by affinity chromatography and tested on a panel of in vitro deiminated proteins. Their ability to bind in vivo deiminated proteins was evaluated by immunoprecipitation, using EBV‐infected cell lines.

Results

Antibodies specific for a peptide corresponding to the EBNA‐1^35–58 sequence containing citrulline in place of arginine (viral citrullinated peptide [VCP]) were detected in 50% of RA sera and in <5% of normal and disease control sera. In addition, affinity‐purified anti‐VCP antibodies from RA sera reacted with filaggrin‐derived citrullinated peptides, with deiminated fibrinogen, and with deiminated recombinant EBNA‐1. Moreover, anti‐VCP antibodies immunoprecipitated, from the lysate of calcium ionophore–stimulated lymphoblastoid cell lines, an 80‐kd band that was reactive with a monoclonal anti–EBNA‐1 antibody and with anti–modified citrulline antibodies.

Conclusion

These data indicate that ACPAs react with a viral deiminated protein and suggest that EBV infection may play a role in the induction of these RA‐specific antibodies.

     

Defective DNA double‐strand break repair in pediatric systemic lupus erythematosus

Objective

Previous reports of cells from patients with systemic lupus erythematosus (SLE) note that repair of single‐strand breaks is delayed, and these lesions may be converted to double‐strand breaks (DSBs) at DNA replication forks. We undertook this study to assess the integrity of DSB recognition, signaling, and repair mechanisms in B lymphoblastoid cell lines derived from patients with pediatric SLE.

Methods

Nine assays were used to interrogate DSB repair and recognition in lymphoblastoid cell lines from patients with pediatric SLE, including the neutral comet assay (NCA), colony survival assay (CSA), irradiation‐induced foci formation for γ‐H2AX and 53BP1 proteins, kinetics of phosphorylation of structural maintenance of chromosomes protein 1 (SMC1), postirradiation bromodeoxyuridine incorporation to evaluate S phase checkpoint integrity, monoubiquitination of Fanconi protein D2, ATM protein expression, and non‐homologous DNA end joining protein expression and function.

Results

Three of the 9 assays revealed abnormal patterns of response to irradiation‐induced DNA damage. The NCA and CSA yielded aberrant results in the majority of SLE lymphoblastoid cell lines. Abnormal prolongation of SMC1 phosphorylation was also noted in 2 of 16 SLE lymphoblastoid cell lines.

Conclusion

Our data suggest that DSB repair is defective in some lymphoblastoid cell lines from pediatric patients with SLE, especially when assessed by both NCA and CSA. Since these studies are nonspecific, further studies of DNA repair and kinetics are indicated to further delineate the underlying pathogenesis of SLE and possibly identify therapeutic targets.

     

Studies of cell‐mediated immune responses to influenza vaccination in systemic lupus erythematosus

Objective

Both antibody and cell‐mediated responses are involved in the defense against influenza. In patients with systemic lupus erythematosus (SLE), a decreased antibody response to subunit influenza vaccine has been demonstrated, but cell‐mediated responses have not yet been assessed. This study was therefore undertaken to assess cell‐mediated responses to influenza vaccination in patients with SLE.

Methods

Fifty‐four patients with SLE and 54 healthy control subjects received subunit influenza vaccine. Peripheral blood mononuclear cells and sera were obtained before and 1 month after vaccination. Cell‐mediated responses to A/H1N1 and A/H3N2 vaccines were evaluated using an interferon‐γ (IFNγ) enzyme‐linked immunospot assay and flow cytometry. Antibody responses were measured using a hemagglutination inhibition test.

Results

Prior to vaccination, patients with SLE had fewer IFNγ spot‐forming cells against A/H1N1 compared with control subjects and a lower frequency of IFNγ‐positive CD8+ T cells. After vaccination, the number of IFNγ spot‐forming cells increased in both patients and control subjects, although the number remained lower in patients. In addition, the frequencies of CD4+ T cells producing tumor necrosis factor and interleukin‐2 were lower in patients after vaccination compared with healthy control subjects. As expected for a subunit vaccine, vaccination did not induce a CD8+ T cell response. For A/H3N2‐specific responses, results were comparable. Diminished cell‐mediated responses to influenza vaccination were associated with the use of prednisone and/or azathioprine. The increase in A/H1N1‐specific and A/H3N2‐specific antibody titers after vaccination was lower in patients compared with control subjects.

Conclusion

In addition to a decreased antibody response, cell‐mediated responses to influenza vaccination are diminished in patients with SLE, which may reflect the effects of the concomitant use of immunosuppressive drugs. This may render these patients more susceptible to (complicated) influenza infections.

     

Neuropsychiatric involvement in pediatric systemic lupus erythematosus

Neuropsychiatric (NP) manifestations are found in approximately 25% of children and adolescents with pediatric SLE (pSLE). In 70% of those, NP involvement will occur within the first year from the time of diagnosis. Headaches (66%), psychosis (36%), cognitive dysfunction (27%) and cerebrovascular disease (24%) are the most common presentations. The support of a psychiatrist is often required. Anti-phospholipid antibodies are associated with distinct NP disease entities and may be implicated in the pathogenesis of several manifestations of NP-pSLE including chorea, cerebrovascular disease and seizures. The role of novel auto-antibodies and imaging modalities is currently explored. The treatment of NP-pSLE is not based on prospective studies; however, an immunosuppressive combination therapy consisting of high doses of prednisone and a second line agent such as cyclophosphamide or azathioprine is commonly suggested for children with NP-pSLE. The role of novel therapies is currently studied. The outcome of children with NP-pSLE is relatively good. The overall survival is 95-97%, 20% of children experience a disease flare during childhood and 25% have evidence of permanent neuropsychiatric damage.     

An unusual pediatric case with neurofibromatosis and systemic lupus erythematosus

Neurofibromatosis type 1 (NF1) is a relatively common autosomal dominant disorder affecting mainly ectodermal and mesodermal tissues. It is well known that patients with NF1 have an increased risk of developing benign and malignant tumors, but its association with autoimmune diseases has been rarely reported. Systemic lupus erythematosus is an autoimmune chronic inflammatory disease that has the potential to affect various organ systems. There are four cases with NF1 and SLE reported in the literature up to date. Here, we report a 9-year-old girl presenting with NF1 and SLE, and to our knowledge, this is the first childhood case in the literature.     

Infliximab‐induced systemic lupus erythematosus

Only six cases of clinical drug‐induced lupus (DIL) following infliximab initiation for rheumatoid arthritis and Crohn's disease are documented dispite studies indicating many patients develop asymptomatic antinuclear ANA (62%) and anti‐dsDNA (15%) antibodies. Our 43‐year‐old female patient with 25 years of RA had active synovitis despite combination sulfasalazine, leflunomide and methotrexate. She received infliximab (3 mg/kg) infusion with rapid resolution of synovitis and then again 2, 6 and 14 weeks later. At 21 weeks she developed acute DIL with typical clinical features and laboratory findings which resolved after 8 weeks of prednisolone treatment. New biologic therapies can not only suppress RA but potentiate clinical DIL.     

Measuring permanent damage in pediatric systemic lupus erythematosus

The survival rates in pediatric systemic lupus erythematosus (pSLE) have improved greatly over recent decades. Increased life expectancy has meant that more children are growing up with the consequences of chronic disease and prolonged therapy. Assessing complications of disease and its therapy becomes an important outcome measure by which to evaluate our therapeutic interventions and appraise quality of life. In this paper we review the development of the Systemic Lupus International Collaborative Clinics (SLICC)/American College of Rheumatology (ACR) Damage Index (SDI) and its application to the pSLE population. We examine the profile of damage in pSLE as identified by the SDI. However we also critically appraise its application and identify potential limitations in the SDI as a measure of permanent disease damage in children. In this paper we put forth suggestions for additional domains addressing pediatric specific issues such as decreased final height and delayed puberty. We also suggest modifications to domains of gonadal failure, diabetes mellitus, cognitive impairment and osteonecrosis in the SDI to make it more reflective of the damage phenomenon observed in pediatrics.     

Chylomicron metabolism is markedly altered in systemic lupus erythematosus

OBJECTIVE: To verify the in vivo status of chylomicron metabolism in systemic lupus erythematosus (SLE) since there is a high incidence of atherosclerosis in this disease and chylomicrons may have an important role in atherogenesis. METHODS: A chylomicron-like emulsion labeled with 14C-cholesteryl esters and 3H-triglycerides was injected intravenously into 10 female patients with inactive SLE and 10 healthy age- and sex-matched control subjects to determine the plasma kinetics of the emulsion lipids from consecutive plasma samples taken at regular intervals for 1 hour. Lipolytic activity was determined in vitro after incubation of the labeled emulsion with postheparin plasma. RESULTS: The decay curves for the emulsion were markedly slowed in SLE. Chylomicron lipolysis, indicated by the fractional clearance rate (FCR) of emulsion 3H-triglyceride, was 2-fold smaller in SLE patients than in controls (mean +/- SD 0.023 +/- 0.011 versus 0.047 +/-0.015 minute(-1); P = 0.010). Chylomicron removal, indicated by emulsion 14C-cholesteryl ester FCR, was 3-fold smaller in SLE patients than in controls (0.007 +/-0.007 versus 0.023 +/- 0.011 minute(-1); P = 0.009). In vitro lipolysis in SLE patients was nearly half that of the controls (mean +/- SD 10,199 +/- 2,959 versus 6,598 +/-2,215; P = 0.014). Higher levels of very-low-density lipoprotein cholesterol and triglycerides and lower levels of high-density lipoprotein cholesterol and apolipoprotein A-I were also observed in the SLE patients. CONCLUSION: SLE patients have disturbances in chylomicron metabolism that are characterized by decreased lipolysis and chylomicron remnant removal from the plasma. This finding, together with other alterations in lipid profiles that were confirmed in the present study, is largely accountable for the accelerated atherosclerotic process of the disease.