Endomyocardial biopsies in the diagnosis of myocardial involvement in systemic lupus erythematosus.

BACKGROUND: Endomyocardial biopsy (EMB) is considered the gold standard for diagnosing myocardial involvement in most inflammatory conditions, including systemic lupus erythematosus (SLE). However, EMBs are rarely performed, and most of the myocardial histopathology reports in SLE consist of postmortem data. We therefore sought to describe the histopathologic findings of contemporary EMBs in SLE performed in clinical practice. METHODS: A retrospective review of histopathology reports from SLE patients who underwent EMB from 1994 to 2017 was performed. A total of 41 SLE patients had cardiac pathology reports. Of these, 11 histopathology reports were EMBs, and the remaining were valvular specimens. RESULTS: A total of 11 SLE EMBs were reviewed. It was found that 45% of the patients had hypertension, 27% had coronary artery disease, 9% had hyperlipidemia, and 36% had end-stage renal disease. None had diabetes or smoked. The mean left ventricular ejection fraction was 37%. On histopathology, 10 had mild interstitial fibrosis, 9 had myocyte hypertrophy, 3 had organized blood clots, and 3 had a mild infiltration of lymphocytes and macrophages without clear evidence of myocarditis. None had vasculitis, endocarditis, ischemia, amyloid deposition, or lamellar or curvilinear inclusions. CONCLUSION: EMBs are rarely performed in SLE. In this case series, nonspecific interstitial fibrosis and myocyte hypertrophy were the most common findings, suggesting EMBs have limited value in the diagnosis of cardiac involvement in SLE and rather rule out competing conditions. These data support the need for diagnostic methods with high sensitivity and specificity for SLE heart disease.

Association between hydroxychloroquine levels and disease activity in a predominantly Hispanic systemic lupus erythematosus cohort.

OBJECTIVES: Hydroxychloroquine (HCQ) is a key therapy in systemic lupus erythematosus (SLE). Medication non-adherence is reported in up to 80% of lupus patients and results in increased morbidity, mortality, and health care utilization. HCQ levels are a sensitive and reliable method to assess medication adherence. Our study evaluated the role of HCQ level measurement in routine clinical care and its association with disease activity in a predominantly Hispanic population. METHODS: SLE patients from the Columbia University Lupus cohort treated with HCQ for ≥ 6 months and reporting medication adherence were included. HCQ levels were measured by whole blood high performance liquid chromatography. Non-adherence was defined as an HCQ level <500 ng/ml. The association between HCQ levels and disease activity measured by Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) was evaluated. RESULTS: One hundred and eight patients were enrolled; the median age was 38 years, 91% were female, and 63% were Hispanic. The median SLEDAI-2K was 4.3 (0-20). Forty-one percent of patients had an HCQ level <500 ng/ml consistent with non-adherence, of which 19% had undetectable levels. A higher SLEDAI-2K score was associated with low HCQ levels (p = 0.003). This association remained significant after adjusting for depression (p = 0.0007). CONCLUSION: HCQ levels < 500 ng/ml were associated with higher disease activity and accounted for 32% of the SLEDAI-2K variability. HCQ blood measurement is a simple and reliable method to evaluate medication adherence in SLE. Reasons for non-adherence (levels < 500 ng/ml) should be further explored and addressed.

Cell-bound complement activation products (CB-CAPs) have high sensitivity and specificity in pediatric-onset systemic lupus erythematosus and correlate with disease activity.

OBJECTIVE: Elevated levels of cell-bound complement activation products (CB-CAPs) (C4d deposition on B lymphocytes (BC4d) and/or erythrocytes (EC4d)) are sensitive and specific in diagnosis and monitoring of adult systemic lupus erythematosus (SLE). Our objective was to evaluate the role of CB-CAPs for diagnosis and monitoring of pediatric-onset SLE (pSLE). METHODS: A prospective cohort study of 28 pSLE and 22 juvenile arthritis patients was conducted. SLE disease activity was determined using a clinical Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) that excluded serologies. Autoantibodies were measured using solid-phase immunoassays, C3 and C4 using immunoturbidimetry, and CB-CAPs using quantitative flow cytometry. Abnormal CB-CAPs were defined as EC4d or BC4d above the 99th percentile for healthy adults (>14 and > 60 net mean fluorescence intensity (MFI), respectively). Performance characteristics of CB-CAPs were assessed using area under the curve (AUC) for receiver operating characteristics. Linear mixed effect models evaluated the correlation between CB-CAPs and clinical SLEDAI over 6 months. RESULTS: BC4d yielded higher AUC (0.91 ± 0.04) than C3 (0.63 ± 0.08) and C4 (0.67 ± 0.08) ( p < 0.05). Abnormal CB-CAPs were 78% sensitive and 86% specific for diagnosis of pSLE (Youden's index = 0.64 ± 0.11). In contrast to BC4d, EC4d levels correlated with clinical SLEDAI ( p < 0.01). CONCLUSION: CB-CAPs (EC4d and BC4d) have higher sensitivity and specificity than low complement in pSLE, and may help with diagnosis of pSLE. EC4d could provide a useful biomarker for disease activity monitoring.

Damage accrual in systemic lupus erythematosus in Dominicans in New York City and the Dominican Republic.

Objectives Hispanics with systemic lupus erythematosus (SLE) in the United States have more severe disease and damage accrual compared with whites. Data on Hispanics of similar ancestry in geographically different locations is limited but essential in defining genetic and environmental factors for SLE. This study evaluates SLE disease burden in two Dominican communities, Washington Heights in New York City (NYC) and Santiago in the Dominican Republic (DR). Methods Disease activity (SLE Disease Activity Index 2000 (SLEDAI-2K)) and damage (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI)) were cross-sectionally measured in 76 Dominican SLE patients from the Columbia University Lupus Cohort in NYC and compared with 75 Dominican SLE patients living in Santiago in the DR. Results Mean (±SD) age was 40 (±14) and 36 (±11) years for NYC and DR patients, respectively. Median disease duration was 8 years. Disease activity was mild in both groups (SLEDAI-2K of 3 in NYC versus 4 in the DR). NYC Dominicans had more discoid lesions, positive anti-dsDNA, and anti-SSB antibodies. Dominicans in the DR used more corticosteroids, had less medical insurance, lower educational level, and were more likely to be unemployed, whereas more Dominicans in NYC smoked. NYC patients had a higher SDI compared with SLE patients in the DR (0.96 versus 0.24, p < 0.0001). Statistical significance was maintained in adjusted analysis (1.26 versus 0.57, p < 0.0001). Conclusion SLE Dominican patients in NYC had a higher SDI than those in the DR. Longitudinal studies are needed to ascertain whether this difference is due to biological, environmental factors, immigration patterns or a survival bias.

Study of Flare Assessment in Systemic Lupus Erythematosus Based on Paper Patients.

OBJECTIVE: To determine the level of agreement of disease flare severity (distinguishing severe, moderate, and mild flare and persistent disease activity) in a large paper-patient exercise involving 988 individual cases of systemic lupus erythematosus. METHODS: A total of 988 individual lupus case histories were assessed by 3 individual physicians. Complete agreement about the degree of flare (or persistent disease activity) was obtained in 451 cases (46%), and these provided the reference standard for the second part of the study. This component used 3 flare activity instruments (the British Isles Lupus Assessment Group [BILAG] 2004, Safety of Estrogens in Lupus Erythematosus National Assessment [SELENA] flare index [SFI] and the revised SELENA flare index [rSFI]). The 451 patient case histories were distributed to 18 pairs of physicians, carefully randomized in a manner designed to ensure a fair case mix and equal distribution of flare according to severity. RESULTS: The 3-physician assessment of flare matched the level of flare using the 3 indices, with 67% for BILAG 2004, 72% for SFI, and 70% for rSFI. The corresponding weighted kappa coefficients for each instrument were 0.82, 0.59, and 0.74, respectively. We undertook a detailed analysis of the discrepant cases and several factors emerged, including a tendency to score moderate flares as severe and persistent activity as flare, especially when the SFI and rSFI instruments were used. Overscoring was also driven by scoring treatment change as flare, even if there were no new or worsening clinical features. CONCLUSION: Given the complexity of assessing lupus flare, we were encouraged by the overall results reported. However, the problem of capturing lupus flare accurately is not completely solved.

The prevalence and determinants of anti-DFS70 autoantibodies in an international inception cohort of systemic lupus erythematosus patients.

Autoantibodies to dense fine speckles 70 (DFS70) are purported to rule out the diagnosis of SLE when they occur in the absence of other SLE-related autoantibodies. This study is the first to report the prevalence of anti-DFS70 in an early, multinational inception SLE cohort and examine demographic, clinical, and autoantibody associations. Patients were enrolled in the Systemic Lupus International Collaborating Clinics (SLICC) inception cohort within 15 months of diagnosis. The association between anti-DFS70 and multiple parameters in 1137 patients was assessed using univariate and multivariate logistic regression. The frequency of anti-DFS70 was 7.1% (95% CI: 5.7-8.8%), while only 1.1% (95% CI: 0.6-1.9%) were monospecific for anti-DFS70. In multivariate analysis, patients with musculoskeletal activity (Odds Ratio (OR) 1.24 [95% CI: 1.10, 1.41]) or with anti-ß2 glycoprotein 1 (OR 2.17 [95% CI: 1.22, 3.87]) were more likely and patients with anti-dsDNA (OR 0.53 [95% CI: 0.31, 0.92]) or anti-SSB/La (OR 0.25 [95% CI: 0.08, 0.81]) were less likely to have anti-DFS70. In this study, the prevalence of anti-DFS70 was higher than the range previously published for adult SLE (7.1 versus 0-2.8%) and was associated with musculoskeletal activity and anti-ß2 glycoprotein 1 autoantibodies. However, 'monospecific' anti-DFS70 autoantibodies were rare (1.1%) and therefore may be helpful to discriminate between ANA-positive healthy individuals and SLE.

Low prevalence of Pneumocystis pneumonia in hospitalized patients with systemic lupus erythematosus: review of a clinical data warehouse.

Objective In the era of powerful immunosuppression, opportunistic infections are an increasing concern in systemic lupus erythematosus. One of the best-studied opportunistic infections is Pneumocystis pneumonia; however, the prevalence of Pneumocystis pneumonia in systemic lupus erythematosus is not clearly defined. This study evaluates the prevalence of Pneumocystis pneumonia in hospitalized systemic lupus erythematosus patients, with a focus on validating the Pneumocystis pneumonia and systemic lupus erythematosus diagnoses with clinical information. Methods This retrospective cohort study evaluates the prevalence of Pneumocystis pneumonia in all systemic lupus erythematosus patients treated at Columbia University Medical Center-New York Presbyterian Hospital between January 2000 and September 2014, using electronic medical record data. Patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and patients with renal transplants (including both early and late post-transplant patients) represented immunocompromised control groups. Patients with systemic lupus erythematosus, Pneumocystis pneumonia, HIV/AIDS, or renal transplant were identified using diagnostic codes from the International Classification of Diseases, Ninth Revision (ICD-9). Results Out of 2013 hospitalized systemic lupus erythematosus patients, nine had presumed Pneumocystis pneumonia, yielding a low prevalence of Pneumocystis pneumonia in systemic lupus erythematosus of 0.45%. Three of the nine Pneumocystis pneumonia cases were patients with concomitant systemic lupus erythematosus and HIV/AIDS. Only one of these nine cases was histologically confirmed as Pneumocystis pneumonia, in a patient with concomitant systemic lupus erythematosus and HIV/AIDS and a CD4 count of 13 cells/mm3. The prevalence of Pneumocystis pneumonia in renal transplant patients and HIV/AIDS patients was 0.61% and 5.98%, respectively. Conclusion Given the reported high rate of adverse effects to trimethoprim-sulfamethoxazole in systemic lupus erythematosus and the low prevalence of Pneumocystis pneumonia in hospitalized systemic lupus erythematosus patients, our data do not substantiate the need for Pneumocystis pneumonia prophylaxis in systemic lupus erythematosus patients, except in those with concurrent HIV/AIDS.

Patient experience in systemic lupus erythematosus: development of novel patient-reported symptom and patient-reported impact measures.

BACKGROUND: Comprehensive assessment of systemic lupus erythematosus (SLE) and its treatment requires patient-reported outcome (PRO) measures to capture impacts and fluctuating symptoms. The objective of this study was to develop PROs, in accordance with the Food and Drug Administration (FDA) PRO Guidance, to assess fluctuations in SLE symptoms and its impact. METHODS: Following independent review board approval, six US rheumatology practices recruited patients with SLE to participate in concept elicitation (CE) interviews, in order to identify important SLE symptoms and their impacts. The SLE Symptom Severity Diary (SSD) and SLE Impact Questionnaire (SIQ) were drafted based on CE interview results and clinician input. The PROs were revised based on patient feedback from cognitive debriefing (CD) interviews, clinician feedback, and a translatability assessment. RESULTS: Forty-one patients completed CE interviews. Commonly-reported symptoms included fatigue (98%), joint pain (93%), and rash (88%). The most frequently reported impact was difficulty with chores/housework (61%). Eighteen patients completed CD interviews. The PROs were considered comprehensive, clear, and relevant.The SSD contains 17 items assessing energy/vitality, joint and muscle pain/stiffness/swelling, flu-like symptoms, cognition, numbness/tingling, skin symptoms and hair loss using an 11-point numeric response scale and a 24-h recall period (with the exception of hair loss). It also evaluates steroid status and dose. The SIQ contains 50 items, uses a 5-point Likert scale and a 7-day recall period, to assess disease impacts including patients' ability to make plans, work, and physical/social/emotional functioning. CONCLUSION: The SSD and SIQ are comprehensive SLE-specific PROs developed in accordance with the FDA PRO Guidance. Following assessment of their measurement properties, they may be useful in clinical studies and clinical practice to measure fluctuations in, and the impact of, symptoms in patients with SLE.

Targeted B cell therapies in the treatment of adult and pediatric systemic lupus erythematosus.

Belimumab (Benlysta) is a fully-humanized monoclonal antibody that inhibits B-lymphocyte stimulator (also known as B cell activating factor) and was approved by the U.S. Federal Drug Administration and European Medicines Evaluation Agency for treatment in adults with autoantibody-positive systemic lupus erythematosus (SLE). Rituximab (Rituxan) is a chimeric anti-CD20 monoclonal antibody targeting B lymphocytes. This review discusses the key findings of the phase III trials in adults with SLE and of real-world use of belimumab and rituximab in the care of both adult and pediatric SLE patients. It highlights the safety profile of belimumab and rituximab and gives insight into the consideration of these therapies for specific SLE disease states. It concludes with a discussion of the current clinical trials investigating B cell therapies in specific SLE disease states and a look to the future, with ongoing clinical trials.

Cardiovascular events prior to or early after diagnosis of systemic lupus erythematosus in the systemic lupus international collaborating clinics cohort.

OBJECTIVE: To describe the frequency of myocardial infarction (MI) prior to the diagnosis of systemic lupus erythematosus (SLE) and within the first 2 years of follow-up. METHODS: The systemic lupus international collaborating clinics (SLICC) atherosclerosis inception cohort enters patients within 15 months of SLE diagnosis. MIs were reported and attributed on a specialised vascular event form. MIs were confirmed by one or more of the following: abnormal ECG, typical or atypical symptoms with ECG abnormalities and elevated enzymes (≥2 times upper limit of normal), or abnormal stress test, echocardiogram, nuclear scan or angiogram. Descriptive statistics were used. RESULTS: 31 of 1848 patients who entered the cohort had an MI. Of those, 23 patients had an MI prior to SLE diagnosis or within the first 2 years of disease. Of the 23 patients studied, 60.9% were female, 78.3% were Caucasian, 8.7% black, 8.7% Hispanic and 4.3% other. The mean age at SLE diagnosis was 52.5±15.0 years. Of the 23 MIs that occurred, 16 MIs occurred at a mean of 6.1±7.0 years prior to diagnosis and 7 occurred within the first 2 years of follow-up. Risk factors associated with early MI in univariate analysis are male sex, Caucasian, older age at diagnosis, hypertension, hypercholesterolaemia, family history of MI and smoking. In multivariate analysis only age (OR=1.06 95% CI 1.03 to 1.09), hypertension (OR=5.01, 95% CI 1.38 to 18.23), hypercholesterolaemia (OR=4.43, 95% CI 1.51 to 12.99) and smoking (OR=7.50, 95% CI 2.38 to 23.57) remained significant risk factors. CONCLUSIONS: In some patients with lupus, MI may develop even before the diagnosis of SLE or shortly thereafter, suggesting that there may be a link between autoimmune inflammation and atherosclerosis.

Sex differences in genomics in lupus: girls with systemic lupus have high interferon gene expression while boys have high levels of tumour necrosis factor-related gene expression.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a chronic disease occurring up to 15 times more frequently in females than males. This bias extends to possible differences in disease flares and response to therapy. This study was initiated to investigate the differences between girls and boys with childhood-onset SLE (cSLE) at the molecular level. METHOD: We analysed the Gene Expression Omnibus National Center for Biotechnology Information (GEO NCBI) microarray data available for 88 girls and 16 boys with treatment-naïve cSLE and compared the results to those from healthy controls. Transcriptional profiles were generated using the platforms of Affymetrix U133A and U133B gene chips and Bioconductor/R programming packages were used to process and compare the data. RESULTS: Girls with cSLE overexpressed an interferon (IFN)-α signature that was absent in boys. Boys with cSLE were observed to overexpress tumour necrosis factor-related genes that were absent in girls. Both boys and girls were observed to overexpress several genes related to granulopoeisis. CONCLUSIONS: Our results suggest a potential application of genomics to differentially predict response to therapy between females and males with SLE.

Effect of belimumab treatment on renal outcomes: results from the phase 3 belimumab clinical trials in patients with SLE.

A pooled post-hoc analysis of the phase 3, randomized, placebo-controlled BLISS trials (1684 patients with active systemic lupus erythematosus (SLE)) was performed to evaluate the effect of belimumab on renal parameters in patients with renal involvement at baseline, and to explore whether belimumab offered additional renal benefit to patients receiving mycophenolate mofetil at baseline. In addition to belimumab or placebo, all patients received standard SLE therapy. Patients with severe active lupus nephritis were excluded from the trials. Over 52 weeks, rates of renal flare, renal remission, renal organ disease improvement (assessed by Safety of Estrogens in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index and British Isles Lupus Assessment Group), proteinuria reduction, grade 3/4 proteinuria, and serologic activity favored belimumab, although the between-group differences in most renal outcomes were not significant. Among the 267 patients with renal involvement at baseline, those receiving mycophenolate mofetil or with serologic activity at baseline had greater renal organ disease improvement with belimumab than with placebo. Limitations of this analysis included the small patient numbers and the post-hoc nature of this pooled analysis. The results suggest that belimumab may offer renal benefit in patients with SLE. Further study is warranted in patients with severe active lupus nephritis.

Association between myasthaenia gravis and systemic lupus erythematosus: three case reports and review of the literature.

The coexistence of systemic lupus erythematosus (SLE) and myasthaenia gravis (MG) has been reported previously. Because of their shared clinical characteristics and autoantibody-mediated pathogenesis, an SLE expert panel decided to include MG as one of the 19 neuropsychiatric SLE syndromes. This study reports a cluster of three cases of SLE/MG overlap from our cohort and a review of the published data concerning this overlap of SLE and MG. A systematic Medline review revealed 13 cases described in eight publications from 1994 to 2009. In summary, 12 of the 16 patients (three from our cohort and 13 from the reported cases) were women with an average age of 34 years. The most common SLE manifestations were polyarthritis (15 out of 16 patients), skin rashes (5/16), serositis (5/16), and cytopaenias (10/16). All of the patients were anti-nuclear antibodies (ANA) positive and 15/16 were anti-dsDNA positive. Proximal muscle weakness was the most frequent MG-related symptom (9/16), while 11/16 patients were anti-acetylcholine receptor (anti-AChR) antibody positive and 9/16 had diagnostic electromyography (EMG). These data suggest that MG should to be included in the differential diagnosis of lupus patients with fatigue and muscular weakness together with inflammatory and drug-induced myopathy.

Frequency of neuro-psychiatric dysfunction in anti-SSA/SSB exposed children with and without neonatal lupus.

Neonatal lupus is a model of passively acquired autoimmunity whereby anti-SSA/Ro-SSB/La antibodies target the fetal heart and neonatal skin in a minority of cases. Since neuro-psychiatric impairment has been reported in humans and mice exposed prenatally to a variety of maternal autoantibodies including anti-Ro/La, this study was initiated to evaluate the potential neurotoxic effects of these specific autoantibodies and the overall frequency of autoimmune diseases, general health, and somatic growth of children with neonatal lupus and their unaffected siblings. In addition to the general health questionnaires maintained on family members enrolled in the Research Registry for Neonatal Lupus (RRNL), specific questionnaires related to neuro-psychiatric development were sent to all mothers whose children (both affected and unaffected) were older than 5 years of age. Controls consisted of healthy friends. Of 121 anti-Ro exposed children meeting the inclusion criteria, information was returned on 104 (33 cardiac manifestations of neonatal lupus, 20 rash, and 51 unaffected siblings) and 22 of the friend controls. The mean age of all of the children was 14.5 years (range 5-39). In total, 42 (40%) of the 104 anti-Ro exposed children were reported to have a neuro-psychiatric disorder, compared with 6 (27%) of the friend controls (p = 0.34). For 8 (24%) of the congenital heart block (CHB) children (6 boys, 2 girls) the mothers reported attention problems. Four, all boys, were on stimulants. Of the rash children, 4 (20%) (2 boys, 2 girls) had attention problems with one boy on Ritalin. Of the unaffected siblings, 9 (18%) (8 boys and 1 girl) had attention problems with 3 boys on stimulants. One (5%) of the control children (a girl) had attention problems, not requiring therapy. There was no statistical difference in attention problems between the groups (p = 0.120). Behavioral problems were present in all groups with no statistical differences noted. The prevalence of depression, anxiety, developmental delays, learning, hearing, and speech problems were not significantly different between groups. In the CHB children, one boy has nephrotic syndrome and one girl has psoriasis. In the rash children, one girl has juvenile rheumatoid arthritis. In the unaffected group there are five children with autoimmune diseases, two with inflammatory bowel diseases (one boy and one girl), one boy has a spondyloarthropathy, one girl has alopecia areata and one young woman has Antiphospholipid syndrome. In the control group one boy has Henoch Schonlein purpura. There were four cases of hypothyroidism, possibly secondary to Hashimoto's thyroiditis, three in boys with CHB and one in a girl with rash. None of the unaffected siblings or controls had hypothyroidism. Parental reporting of neuro-psychiatric abnormalities was high in anti-Ro exposed children regardless of the neonatal lupus manifestation. However, medication use was limited and although the frequency of this reporting was greater than friend controls, it did not reach significance.

Spectrum and progression of conduction abnormalities in infants born to mothers with anti-SSA/Ro-SSB/La antibodies.

The classic cardiac manifestation of neonatal lupus is congenital heart block, attributed to antibody-mediated inflammation and subsequent fibrosis of the atrioventricular (AV) node. In considering the pathologic process of injury it may be that tissue damage results in a range of conduction abnormalities. Identification of less-advanced degrees of block or of fibrosis around the AV node without any conduction abnormality on EKG would support this pathologic model, and serve as a potential marker for treatment if the conduction defect could be shown to progress. To ascertain the spectrum of arrhythmias associated with maternal anti-SSA/Ro-SSB/La antibodies, records of all children enrolled in the Research Registry for Neonatal Lupus were reviewed. Of 187 children with congenital heart block whose mothers have anti-SSA/Ro-SSB/La antibodies, nine had a prolonged PR interval on EKG at birth, four of whom progressed to more advanced AV block. A child whose younger sibling had third degree block was diagnosed with first degree block at age 10 years at the time of surgery for a broken wrist. Two children diagnosed in utero with second degree block were treated with dexamethasone and reverted to normal sinus rhythm by birth, but ultimately progressed to third degree block. Four children had second degree block at birth: of these, two progressed to third degree block. Sinus bradycardia (< 100 bpm) was present in three (3.8%) of 78 fetuses for whom atrial rates were recorded by echocardiogram. Of 40 neonates for whom EKGs were available, the mean atrial rate was 137+/-20 bpm (range 75-200). These data have important research and clinical implications. In contrast to the AV node, permanent sinoatrial nodal involvement is not clinically apparent. Perhaps many fetuses sustain mild inflammation, but resolution is variable, as suggested by the presence of incomplete AV block. Since subsequent progression of less-advanced degrees of block can occur, an EKG should be performed on all infants born to mothers with anti-SSA/Ro-SSB/La antibodies.

Interaction between 52 kDa SSA/Ro and deubiquitinating enzyme UnpEL: a clue to function.

The detection of isolated heart block in utero strongly predicts the presence of maternal autoantibodies reactive with 52 kDa SSA/Ro. The mechanisms that underlie this observation may be elucidated by defining the function of the target antigen. The initial approach was to identify proteins interactive with 52Ro using transcriptional activity in the yeast 2-hybrid system. A cDNA library was constructed using RNA isolated from human fetal hearts (12-23 weeks) and cloned into the HybriZAP vector encoding the activation domain of GAL4(AD) as target. Approximately 7 x 10(6) cDNAs were cotransformed with the bait into YRG-2. Plasmids from five interactive colonies were sequenced and three identified as the specific human deubiquitinating enzyme, UnpEL. UnpEL did not interact with bait plasmid encoding 52 beta, an alternative leucine zipper-minus form of 52 kDa SSA/Ro which is maximally expressed in fetal life. The mammalian 2-hybrid assay confirmed the interaction between full-length 52Ro and UnpEL. Further support for a biologic interaction was the marked redistribution in cellular localization of UnpEL following cotransfection of the two proteins into cultured human cardiocytes, human renal carcinoma cells (293 cells), and monkey kidney fibroblasts (COS-1). In conclusion, the interaction of full-length 52Ro and UnpEL implies that the former may also be involved in the ubiquitin pathway, an observation of particular interest since 52Ro contains a RING finger domain, a motif common to several recently reported proteins involved in modulating ubiquitination. The absence of an interaction with 52 beta raises the consideration that regulation of protein ubiquitination might differ in fetal life.

Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of TNF-alpha by macrophages.

Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16-24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-alpha from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.