W. Joseph McCune

A phase II, randomized, double-blind, placebo-controlled, dose-ranging study of belimumab in patients with active systemic lupus erythematosus.

OBJECTIVE To assess the safety, tolerability, biologic activity, and efficacy of belimumab in combination with standard of care therapy (SOC) in patients with active systemic lupus erythematosus (SLE). METHODS Patients with a Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score >/=4 (n = 449) were randomly assigned to belimumab (1, 4, or 10 mg/kg) or placebo in a 52-week study. Coprimary end points were the percent change in the SELENA-SLEDAI score at week 24 and the time to first SLE flare. RESULTS Significant differences between the treatment and placebo groups were not attained for either primary end point, and no dose response was observed. Reductions in SELENA-SLEDAI scores from baseline were 19.5% in the combined belimumab group versus 17.2% in the placebo group. The median time to first SLE flare was 67 days in the combined belimumab group versus 83 days in the placebo group. However, the median time to first SLE flare during weeks 24-52 was significantly longer with belimumab treatment (154 versus 108 days; P = 0.0361). In the subgroup (71.5%) of serologically active patients (antinuclear antibody titer >/=1:80 and/or anti-double-stranded DNA [anti-dsDNA] >/=30 IU/ml), belimumab treatment resulted in significantly better responses at week 52 than placebo for SELENA-SLEDAI score (-28.8% versus -14.2%; P = 0.0435), physicians global assessment (-32.7% versus -10.7%; P = 0.0011), and Short Form 36 physical component score (+3.0 versus +1.2 points; P = 0.0410). Treatment with belimumab resulted in a 63-71% reduction of naive, activated, and plasmacytoid CD20+ B cells, and a 29.4% reduction in anti-dsDNA titers (P = 0.0017) by week 52. The rates of adverse events and serious adverse events were similar in the belimumab and placebo groups. CONCLUSION Belimumab was biologically active and well tolerated. The effect of belimumab on the reduction of SLE disease activity or flares was not significant. However, serologically active SLE patients responded significantly better to belimumab therapy plus SOC than to SOC alone.

Association of plasma B lymphocyte stimulator levels and disease activity in systemic lupus erythematosus

OBJECTIVE To determine the association of plasma B lymphocyte stimulator (BLyS) levels, immunosuppressive therapy, and other clinical parameters with disease activity in systemic lupus erythematosus (SLE). METHODS Two hundred forty-five SLE patients were evaluated prospectively over a 2-year period at 4 centers. Assessments were performed every 3-6 months. Univariate analysis was used to determine the association among the Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score, serum anti-double-stranded DNA (anti-dsDNA), and plasma BLyS levels. A multivariate repeated-measures model incorporating immunosuppressive therapy was utilized. RESULTS Ninety-two percent of the patients were female. Sixty-seven percent were white, 31% African American, and 2% Asian (all of these groups may include Hispanic). Mean values at baseline were as follows: age 41.5 years, disease duration 8.1 years, SELENA-SLEDAI 3.3 (median 2, range 0-18), BLyS 5.57 ng/ml, IgG 1,439 mg/dl, C3 104.4 mg/dl, and C4 21.3 mg/dl; among those positive for anti-dsDNA, the median titer was 1:40 (range 1:10-1:1,280). Univariate analysis showed that plasma BLyS levels were associated with anti-dsDNA titers (P = 0.0465) and SELENA-SLEDAI scores (P = 0.0002). In multivariate analyses, a greater increase in the SELENA-SLEDAI score from the previous visit was associated with higher BLyS levels at the previous visit (P = 0.0042) and with a greater increase in the BLyS level from the previous visit (P = 0.0007). CONCLUSION The findings of association between a greater increase in the BLyS level from the previous visit and a greater increase in the SELENA-SLEDAI score at the subsequent visit, and between an elevated BLyS level at the previous visit and a greater SELENA-SLEDAI score at the subsequent visit, demonstrate a relationship between circulating BLyS levels and SLE disease activity. These results lend support to the notion that BLyS is a candidate for therapeutic targeting in SLE.

A Distinct Subset of Proinflammatory Neutrophils Isolated from Patients with Systemic Lupus Erythematosus Induces Vascular Damage and Synthesizes Type I IFNs

Neutrophil-specific genes are abundant in PBMC microarrays from lupus patients because of the presence of low-density granulocytes (LDGs) in mononuclear cell fractions. The functionality and pathogenicity of these LDGs have not been characterized. We developed a technique to purify LDGs from lupus PBMCs and assessed their phenotype, function, and potential role in disease pathogenesis. LDGs, their autologous lupus neutrophils, and healthy control neutrophils were compared with regard to their microbicidal and phagocytic capacities, generation of reactive oxygen species, activation status, inflammatory cytokine profile, and type I IFN expression and signatures. The capacity of LDGs to kill endothelial cells and their antiangiogenic potential were also assessed. LDGs display an activated phenotype, secrete increased levels of type I IFNs, TNF-α, and IFN-γ, but show impaired phagocytic potential. LDGs induce significant endothelial cell cytotoxicity and synthesize sufficient levels of type I IFNs to disrupt the capacity of endothelial progenitor cells to differentiate into mature endothelial cells. LDG depletion restores the functional capacity of endothelial progenitor cells. We conclude that lupus LDGs are proinflammatory and display pathogenic features, including the capacity to synthesize type I IFNs. They may play an important dual role in premature cardiovascular disease development in systemic lupus erythematosus by simultaneously mediating enhanced vascular damage and inhibiting vascular repair.

Clinical and Immunologic Effects of Monthly Administration of Intravenous Cyclophosphamide in Severe Systemic Lupus Erythematosus

Severe systemic lupus erythematosus affecting the kidney or central nervous system may lead to organ failure or death despite treatment with high doses of corticosteroids. To evaluate the clinical and immunologic effects of intravenous cyclophosphamide in this setting, we treated nine patients with monthly intravenous infusions of cyclophosphamide for six months. A comparison of characteristics at entry and follow-up revealed improvements (by paired t-test) in creatinine clearance (66 vs. 96 ml per minute, P less than 0.001); 24-hour urinary protein level (4.11 vs. 0.90 g, P less than 0.05), Farr anti-DNA titer (43 vs. 8.5 percent, P less than 0.01); complement components C3 (894 vs. 1150 mg per liter, P less than 0.05), C4 (154 vs. 222 mg per liter, P less than 0.05), and total complement activity (CH50) (88.7 vs. 113.4 IU, P less than 0.05); and Westergren erythrocyte sedimentation rate (60.2 vs. 34.4 mm per hour, P less than 0.0005). Other manifestations of lupus improved markedly in most cases, despite a reduction in the mean daily dose of prednisone, from 45 mg at entry to 17 mg at follow-up (P less than 0.01). The numbers of lymphocytes positive for T3, T4, T8, and B1 declined progressively during treatment. At follow-up, persistent decreases were observed in the T-lymphocyte subsets, whereas the absolute number of B lymphocytes had returned to levels near base line. T-cell proliferative responses at follow-up were not significantly different from entry values, except that the response to mitogenic anti-T11 (CD2) antibodies was decreased (P less than 0.01). Our data indicate that monthly intravenous administration of cyclophosphamide was associated with a substantial amelioration of severe systemic lupus, in conjunction with discrete changes in T-lymphocyte markers and T-cell function. This was a preliminary, uncontrolled study, but the results warrant further investigation of this form of treatment.

Decreased ras-mitogen-activated protein kinase signaling may cause DNA hypomethylation in T lymphocytes from lupus patients

OBJECTIVE Previous studies have shown that inhibiting T cell DNA methylation causes a lupus-like disease by modifying gene expression. T cells from patients with lupus exhibit diminished levels of DNA methyltransferase (MTase) enzyme activity, hypomethylated DNA, and changes in gene expression similar to those exhibited by T cells treated with methylation inhibitors, suggesting that DNA hypomethylation may contribute to human lupus. Since it is known that DNA MTase levels are regulated by the ras-mitogen-activated protein kinase (MAPK) pathway, this study sought to determine whether decreased ras-MAPK signaling could account for the DNA hypomethylation in lupus T cells. METHODS DNA MTase messenger RNA (mRNA) from lupus patients and from healthy controls was quantitated by Northern analysis, and ras-MAPK signaling was determined by immunoblotting with antibodies to the activated forms of extracellular receptor-associated kinase (ERK). Results were compared with those in T cells in which ras-MAPK signaling was inhibited with a soluble inhibitor of MAPK ERK I (MEK1). RESULTS T cells from patients with active lupus had diminished DNA MTase mRNA levels and decreased signaling through the ras-MAPK pathway. Inhibiting signaling through the ras-MAPK pathway with the MEK1 inhibitor decreased DNA MTase mRNA and enzyme activity to the levels seen in lupus T cells, and resulted in DNA hypomethylation resembling that seen in lupus T cells. CONCLUSION These results suggest that a decrease in signaling through the ras-MAPK pathway may be responsible for the decreased MTase activity and DNA hypomethylation in patients with lupus.

Brief Communication: High Incidence of Venous Thrombotic Events among Patients with Wegener Granulomatosis: The Wegener's Clinical Occurrence of Thrombosis (WeCLOT) Study

Context Are patients with Wegener granulomatosis at increased risk for venous thrombotic events (VTEs)? Contribution This prospective observational study found 16 VTEs in 167 patients with Wegener granulomatosis who had no history of VTE. The incidence of VTE was 7 per 100 person-years of follow-up. Implications Patients with Wegener granulomatosis probably have an increased risk for VTE compared with healthy populations who have less than 1 VTE per 100 person-years offollow-up. The Editors Wegener granulomatosis is characterized by inflammation of small- and medium-sized vessels and granulomatous inflammation of various organs (1, 2). The involvement of the venous system in Wegener granulomatosis has received little attention in the past, with only a few reported cases of venous thrombosis (3-5), and textbooks and review articles do not mention an increased risk for venous thrombotic events (VTEs) (1, 6, 7). Early in the enrollment phase of a multicenter treatment trial for Wegener granulomatosis (8-10), several patients had VTEs, including both deep venous thromboses and pulmonary emboli. This observation led to our investigation of VTE incidence in patients with Wegener granulomatosis. Methods Patients and Visit Schedule The Wegeners Granulomatosis Etanercept Trial (WGET) is a multicenter, randomized, double-blind, placebo-controlled study of the efficacy of etanercept, 25 mg subcutaneously twice weekly, in addition to conventional immunosuppressive therapy with glucocorticoids and either methotrexate or cyclophosphamide. Details of the trial design and study results have been published (8, 10). All patients fulfilled the modified American College of Rheumatology Classification Criteria for Wegener granulomatosis, had no history of either exposure to inhibitors of tumor necrosis factor- or a malignant condition, and had no evidence of active infection upon enrollment (8). All patients in WGET were enrolled and randomly assigned to either the active experimental medication or placebo during a period of active vasculitis (flare). Patients were evaluated at study visits every 3 months. Data collection included a full interim medical history with determination of Wegener granulomatosis disease activity, physical examination, laboratory studies, and assessment and review of adverse events. We measured Wegener granulomatosis disease activity by using the Birmingham Vasculitis Activity Score for Wegeners Granulomatosis (BVAS/WG) (11), which considers all manifestations of active disease present during the 28-day period before the date of assessment. A score of 1 or greater indicates active disease, and a score of 0 indicates remission. Patients were required to have a score of 3 or greater to be enrolled in the trial. Investigators measured cumulative disease damage with the Vasculitis Damage Index (12). Severe disease was defined as having a life- or organ-threatening manifestation; other patients were considered to have limited disease (8). The patients who we observed for incidence of VTEs included all 180 patients enrolled in WGET. Details of the baseline demographic and clinical characteristics of this study cohort have been published (9). Diagnosis and Documentation of VTEs All VTEs in WGET were considered serious adverse events necessitating a separate written report documenting the event and outcome (8). A patient was considered to have had a VTE if the event was clinically apparent and was confirmed by diagnostic studies. Clinical evidence of VTEs included edematous or painful limbs, dyspnea, hypoxemia, chest pain, hemoptysis, or other features of deep venous thrombosis or pulmonary embolism. Diagnostic confirmation included results of vascular ultrasonography, impedance plethysmography, ventilationperfusion scanning, computed tomographic angiography, spiral computed tomograpy, venography, or angiography. Investigators collected detailed clinical data on VTEs on all patients for all events that occurred before and during WGET. A study physician completed a separate standardized thrombosis event form for each VTE on the basis of information obtained from patients, nonstudy physicians, and medical records review. The form included the date of event, clinical details of event, diagnostic test results, and determination of Wegener granulomatosis disease status at the time of event. We excluded thromboses of hemodialysis vascular accesses from these analyses. For our investigation of VTE incidence, the observation period started with the date of enrollment of the first patient (9 June 2000) and ended 3 months after the final patient was enrolled (31 December 2002). Statistical Analyses We evaluated differences among patient characteristics in the Wegener granulomatosis cohort at the start of the observation period with the Wilcoxon rank-sum test for continuous variables and with either chi-square or Fisher exact tests for categorical variables (SAS, version 8.0, SAS Institute, Inc., Cary, North Carolina). We calculated the incidence rate and 95% CIs for VTEs by using Stata, version 8.0 (cii command) (Stata Corp., College Station, Texas). The cumulative incidence curve is based on KaplanMeier estimates. Role of the Funding Sources The National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases, the U.S. Food and Drug Administration Office of Orphan Products, and the Amgen Corporation supported this study. The Amgen Corporation provided the data on the incidence of VTEs among patients with rheumatoid arthritis. The funding sources had no role in the design, conduct, or reporting of the study or in the decision to submit the paper for publication. Results Patients We included data from all 180 study patients enrolled in WGET in our study. Table 1 outlines key demographic characteristics and clinic data for the entire cohort and the VTE subgroups. Table 1. Baseline Demographic Characteristics and Clinical Data of Full Study Cohort and Venous Thrombotic Event Subgroups within Wegener Granulomatosis Cohort Incidence Rates of VTE in Wegener Granulomatosis and Comparison Groups At the end of the observation period, 29 of 180 patients (16%) with Wegener granulomatosis had had a VTE at some time: 13 (7.2%) had a history of VTE before WGET enrollment and 16 (8.9%) had first-time VTEs during WGET. The 16 new VTEs among the 167 patients with no history of VTE occurred over 228 person-years of observation, yielding an incidence rate of 7.0 per 100 person-years (95% CI, 4.0 to 11.4). The rates of VTEs did not differ between the etanercept and placebo groups. Clinical Characteristics of Patients with VTEs Appendix Tables 1 and 2 outline the clinical details of all VTEs among the Wegener granulomatosis study sample during and before WGET, respectively. The median time from WGET enrollment (active disease) to VTE in patients who experienced an event was 2.07 months (range, 0.07 to 21.13 months). The Figure shows the time to first VTE for the Wegener granulomatosis group. No participant had more than 1 VTE during WGET. Figure. Time to first venous thrombotic event (VTE) among patients with Wegener granulomatosis. Ten of 16 patients (63%) had active Wegener granulomatosis at the time of the event during WGET. In addition, 11 of the 16 patients (69%) were found to have active Wegener granulomatosis on the study visit before the event, including 3 of the 7 patients whose Wegener granulomatosis was not active at the time of the event. Visits for these 3 patients occurred 14, 33, and 49 days, respectively, before the event. Thus, for 13 of 16 patients (81%) who had VTEs during WGET, Wegener granulomatosis was active at the time of the event or within 2 months before the event. Before WGET enrollment, 18 VTEs occurred among 13 patients. Information on Wegener granulomatosis disease status was available for 12 of 13 first VTEs: Wegener granulomatosis was active in 10 of 12 cases (83%) at the time of the event. Seven of the 13 first VTEs occurred within the 3 months before WGET randomization, including 3 VTEs occurring less than 2 weeks before randomization. We excluded these 7 events from prospective calculation of incident VTE. There were few differences between the 16 patients who had VTE during WGET and the 151 WGET participants who had no history of VTE (Table 1). Compared with participants who did not have an event, those who had a VTE were older at baseline (mean age, 57.5 years vs. 48.6 years; P= 0.039). Aspirin use did not differ between patients with or without VTEs (2 of 16 patients vs. 14 of 151 patients; P> 0.2). Length of hospitalization (4.5 days vs. 6.0 days; P> 0.2) and the proportion of patients hospitalized (50.0% vs. 34.4%, P> 0.2) also did not differ between patients with VTE during WGET and those without a VTE. Discussion To our knowledge, our study is the first to investigate the incidence of VTE in Wegener granulomatosis using a large, well-characterized study cohort and to identify deep venous thrombosis as an important clinical feature of Wegener granulomatosis. Most VTEs in the WGET occurred either during periods of unequivocally active disease or within 2 months of a documented disease flare. Similarly, most VTEs that occurred before the start of the WGET observation period were also associated with active vasculitis. These results suggest that the increased risk for thrombosis bears an important relationship to disease activity in Wegener granulomatosis. Comparison against other groups of patients with VTEs is helpful to appreciate the magnitude of the increased incidence of VTEs in Wegener granulomatosis (Table 2). In a healthy, male, Swedish population, 65 VTEs occurred over 30 years of follow-up, totaling 21007 observation-years and resulting in an incidence rate of first VTE of 0.31 per 100 person-years (CI, 0.4 to 0.4 per 100 person-years) (13). Comparison with this group is relevant because the age of the sample was similar to that of the WGET cohort; the cli

Biologic activity and safety of belimumab, a neutralizing anti-B-lymphocyte stimulator (BLyS) monoclonal antibody: a phase I trial in patients with systemic lupus erythematosus.

IntroductionThis trial evaluated the safety, biologic activity, and pharmacokinetics of belimumab, a fully human monoclonal antibody that inhibits the biologic activity of the soluble form of the essential B-cell survival factor B-lymphocyte stimulator (BLyS) in patients with systemic lupus erythematosus (SLE).MethodsSeventy patients with mild-to-moderate SLE were enrolled in a phase I, double-blind, randomized study and treated with placebo (n = 13) or belimumab (n = 57) at four different doses (1.0, 4.0, 10, and 20 mg/kg) as a single infusion or two infusions 21 days apart. Patients were followed for 84 to 105 days to assess adverse events, pharmacokinetics, peripheral blood B-cell counts, serology, and SLE disease activity. Data from the study were summarized using descriptive statistics. χ2 type tests were used to analyze discrete variables. The Kruskal-Wallis test, the Wilcoxon test, and the analysis of covariance were used to analyze the continuous variables, as appropriate. The analysis was performed on all randomized patients who received study agent.ResultsThe incidences of adverse events and laboratory abnormalities were similar among the belimumab and placebo groups. Belimumab pharmacokinetics were linear across the 1.0 to 20 mg/kg dose range. Long terminal elimination half-life (8.5 to 14.1 days), slow clearance (7 ml/day per kg), and small volume of distribution (69 to 112 ml/kg) were consistent with a fully human antibody. Significant reductions in median percentages of CD20+ B cells were observed in patients treated with a single dose of belimumab versus placebo (day 42: P = 0.0042; and day 84: P = 0.0036) and in patients treated with two doses of belimumab versus placebo (day 105: P = 0.0305). SLE disease activity did not change after one or two doses of belimumab.ConclusionsBelimumab was well tolerated and reduced peripheral B-cell levels in SLE patients. These data support further studies of belimumab in autoimmune disorders.Trial RegistrationNCT00657007 [clinicaltrials.gov].

Antiproteinase 3 Antineutrophil Cytoplasmic Antibodies and Disease Activity in Wegener Granulomatosis

Context Most patients with Wegener granulomatosis have antineutrophil cytoplasmic antibodies (ANCA). The role of ANCA testing in monitoring response to treatment is controversial. Contribution Using data from a large treatment trial, the authors found little association between disease activity and ANCA levels. Decreases in ANCA levels were not associated with remission, and increases were not associated with relapse. Caution Because follow-up duration differed by patient, standard measures of ANCA accuracy, such as sensitivity and specificity for detecting remission and relapse, could not be calculated. Implication Serial ANCA testing should not be used to monitor disease activity or to guide decisions about immunosuppressive treatment in patients with Wegener granulomatosis. The Editors Wegener granulomatosis is characterized by necrotizing granulomatous inflammation and vasculitis, most commonly affecting the respiratory tract and kidneys (1, 2). Remission can be induced in most patients by using glucocorticoids with cyclophosphamide or methotrexate (17), but most patients have relapse when immunosuppression is reduced or withdrawn (1, 2, 6, 8). Consequently, patients experience substantial illness and damage from both the disease and treatment toxicity (1, 9). Accurate assessment of disease activity and prediction of relapse remain the biggest challenges in management of Wegener granulomatosis (10). Most patients with Wegener granulomatosis have antineutrophil cytoplasmic antibodies (ANCA), which produce a cytoplasmic immunofluorescence pattern on ethanol-fixed neutrophils and react with the neutrophil serine protease proteinase 3 (PR3) (1115). Proteinase 3 is synthesized as a proenzyme (pro-PR3) containing an amino-terminal activation dipeptide that preserves PR3 in an inactive state (16). Subsequent cleavage of this dipeptide allows PR3 to assume its active enzyme conformation (mature-PR3) (16). The diagnostic value of ANCA is well established (17, 18); however, the role of serial ANCA measurements during follow-up and their utility in guiding treatment remain controversial (10, 19, 20). A recent study indicated that in individual patients with Wegener granulomatosis, ANCA against pro-PR3 had a stronger correlation with disease activity than did ANCA against mature-PR3 (21). Therefore, we sought to determine whether pro-PR3ANCA levels correlate more strongly with disease activity than do mature-PR3ANCA levels, whether a decrease in pro- or mature-PR3ANCA levels during remission-induction therapy is associated with a shorter time to sustained remission, and whether an increase in pro- or mature-PR3ANCA levels is associated with relapse. Methods This prospective study was done in the context of the WGET (Wegener Granulomatosis Etanercept Trial) (6, 22, 23), a randomized, placebo-controlled trial that evaluated etanercept for maintenance of remission in 180 patients with Wegener granulomatosis at 8 centers across the United States (Appendix 1). All patients met at least 2 of the 5 modified American College of Rheumatology criteria for classification of Wegener granulomatosis and had active disease within 28 days before enrollment and a Birmingham Vasculitis Activity Score for Wegener granulomatosis (BVAS/WG) of at least 3 (22, 24). Follow-up evaluations were done at baseline, after 6 and 12 weeks, and then every 3 months until the end of the trial. Two additional evaluations took place at 3 and 6 months after the end of the trial. During each visit, disease activity was measured by using the BVAS/WG, and serum samples were obtained, frozen, and stored at 80 C. Treatment Patients were treated in a protocol-defined manner with etanercept or placebo in addition to standard therapies. Patients with severe Wegener granulomatosis (life- or organ-threatening disease) received cyclophosphamide and glucocorticoids at enrollment (22, 24). Those with limited (that is, nonsevere) Wegener granulomatosis received methotrexate and glucocorticoids. Medication dosages were tapered according to protocol once disease activity was controlled (6, 22). Assessment of Disease Activity and Definitions of Sustained Remission and Relapse Disease activity was measured by using the BVAS/WG (24). This index considers all manifestations of active disease during the 28 days preceding the date of assessment. A BVAS/WG of 1 or greater is considered active disease, and a BVAS/WG of 0 indicates remission (24). Our analyses focused on first sustained remission and first relapse. Sustained remission was defined as a BVAS/WG of 0 for at least 6 months (6, 22). The PR3-ANCA level at the visit in the middle of this period was considered the PR3-ANCA level at sustained remission. Disease relapse was defined as an increase of at least 1 point in the BVAS/WG in patients who had sustained remission. ANCA Detection Methods A standard immunofluorescence assay and direct enzyme-linked immunosorbent assays (ELISAs) for PR3-ANCA and ANCA against myeloperoxidase were done as described elsewhere (25). Capture ELISA was used to measure PR3-ANCA (2527); levels are expressed as net absorbance, and a level of 0.10 or greater is considered positive (26, 27). The intra-assay and interassay coefficients of variation are 9% and 31%, respectively, for pro-PR3 capture ELISA, and 6% and 28%, respectively, for mature-PR3 capture ELISA. All baseline serum samples were screened at first thaw by using all methods. Patients whose baseline samples tested positive for perinuclear-staining ANCA or ANCA against myeloperoxidase (n= 24) were excluded from further analyses because of substantial differences in disease phenotype between patients positive for ANCA against myeloperoxidase and those positive for PR3-ANCA. In addition, the number of patients who were positive for ANCA against myeloperoxidase was too small for meaningful longitudinal analysis (5, 28, 29). Subsequently, all serum samples were tested for mature- and pro-PR3ANCA in parallel by using capture ELISA at first thaw (except for the baseline samples, which were retested at second thaw). To minimize variability, all serum samples from an individual patient were run at once in the same plate and the same lots of all reagents were used for all assays. Laboratory personnel were blinded to the clinical data. Increase and Decrease of PR3-ANCA Levels We defined an increase in PR3-ANCA levels a priori as an increase of at least 100% in the net absorbance over 6 months. An absolute increase of at least 0.4 was also required to ensure that small elevations were above the intra-assay coefficient of variation. We classified a negative-to-positive conversion of PR3-ANCA status as an increase only if the absolute increase was at least 0.4. Because 6 months corresponded to 3 clinical visits (except for the initial 6 months after enrollment, when it corresponded to 4 clinical visits), we compared the PR3-ANCA levels at each visit with those from the previous 2 to determine whether the criteria for increase were fulfilled. We first looked for an increase in PR3-ANCA 9 months after enrollment (the fourth visit), because that was the first time that a patient could meet the definition of sustained remission. Thus, the PR3-ANCA level at the fourth visit was compared with the levels at the third and second visits after enrollment. No increase in PR3-ANCA before the fourth visit after enrollment was analyzed (Appendix Figure 1). Appendix Figure 1. Diagram of the initial 4 clinical visits. The fourth clinical visit was the first point at which a patient could meet the study definition of sustained remission (SR) (a Birmingham Vasculitis Activity Score for Wegener granulomatosis [BVAS/WG] of 0 for 6 months) and was the first time we looked for an increase in proteinase 3 (PR3) antineutrophil cytoplasmic antibody (ANCA) levels. We compared the net absorbance of PR3-ANCA at this visit with that of the previous 2 visits or the previous 6 months (curved lines). The same comparison was done at every subsequent visit. We defined a decrease in PR3-ANCA levels during the initial 6 months of follow-up as a decline of at least 50% in the net absorbance with an absolute decrease of at least 0.4; for values between 0.1 and 0.4, the capture ELISA needed to yield negative results. Statistical Analysis Descriptive data are summarized as mean (SD), median (interquartile range), or percentages. Groups were compared by using the t test (or the rank-sum test) or the chi-square test (or Fisher exact test), with 95% CIs calculated as appropriate. A P value less than 0.05 was considered statistically significant. We performed unadjusted and adjusted analyses. The adjusting variables were age, sex, disease severity (severe vs. limited Wegener granulomatosis), treatment group (etanercept vs. placebo), disease duration, baseline BVAS/WG, and clinical center (see Appendix 2 for additional details). PR3-ANCA Levels and Disease Activity The cross-sectional and longitudinal associations between the BVAS/WG and the levels of mature- and pro-PR3ANCA were estimated by using random-effect models. We constructed the models with BVAS/WG as the dependent variable and included a random effect for each patient (random intercept) and 2 terms for PR3-ANCA levelsone term for the value of PR3-ANCA level at baseline and the other for the change in PR3-ANCA level from baseline to time t. To assess the magnitude of the association between PR3-ANCA levels and the BVAS/WG, we estimated the relative reduction in the residual variance by comparing the residual variance of the model that included a random effect for each patient and 2 terms for PR3-ANCA levels with the residual variance of a model that only included the random effect for each patient. These analyses included only observations in which both BVAS/WG and PR3-ANCA level were available (9% of the observations had missing PR3-ANCA information). For patients who achieved sustained remission, the mature- and pro-PR3ANCA level

Population-Based Incidence and Prevalence of Systemic Lupus Erythematosus: The Michigan Lupus Epidemiology and Surveillance Program

OBJECTIVE To estimate the incidence and prevalence of systemic lupus erythematosus (SLE) in a sociodemographically diverse southeastern Michigan source population of 2.4 million people. METHODS SLE cases fulfilling the American College of Rheumatology classification criteria (primary case definition) or meeting rheumatologist-judged SLE criteria (secondary definition) and residing in Wayne or Washtenaw Counties during 2002-2004 were included. Case finding was performed from 6 source types, including hospitals and private specialists. Age-standardized rates were computed, and capture-recapture was performed to estimate underascertainment of cases. RESULTS The overall age-adjusted incidence and prevalence (ACR definition) per 100,000 persons were 5.5 (95% confidence interval [95% CI] 5.0-6.1) and 72.8 (95% CI 70.8-74.8). Among females, the incidence was 9.3 per 100,000 persons and the prevalence was 128.7 per 100,000 persons. Only 7 cases were estimated to have been missed by capture-recapture, adjustment for which did not materially affect the rates. SLE prevalence was 2.3-fold higher in black persons than in white persons, and 10-fold higher in females than in males. Among incident cases, the mean ± SD age at diagnosis was 39.3 ± 16.6 years. Black SLE patients had a higher proportion of renal disease and end-stage renal disease (ESRD) (40.5% and 15.3%, respectively) as compared to white SLE patients (18.8% and 4.5%, respectively). Black patients with renal disease were diagnosed as having SLE at younger age than white patients with renal disease (mean ± SD 34.4 ± 14.9 years versus 41.9 ± 21.3 years; P = 0.05). CONCLUSION SLE prevalence was higher than has been described in most other population-based studies and reached 1 in 537 among black female persons. There were substantial racial disparities in the burden of SLE, with black patients experiencing earlier age at diagnosis, >2-fold increases in SLE incidence and prevalence, and increased proportions of renal disease and progression to ESRD as compared to white patients.

Thrombosis in patients with connective tissue diseases treated with specific cyclooxygenase 2 inhibitors: A report of four cases

Specific inhibitors of cyclooxygenase 2 (COX-2) have been approved for the treatment of osteoarthritis and rheumatoid arthritis. Unlike nonsteroidal anti-inflammatory drugs, specific COX-2 inhibitors do not inhibit platelet activation. However, these agents significantly reduce systemic production of prostacyclin. As a result, theoretical concerns have been raised that specific COX-2 inhibitors could shift the hemostatic balance toward a prothrombotic state. Patients with connective tissue diseases (CTD), who may be predisposed to vasculopathy and thrombosis, often have arthritis or pain syndromes requiring treatment with antiinflammatory agents. Herein we describe 4 patients with CTD who developed ischemic complications after receiving celecoxib. All patients had a history of Raynauds phenomenon, as well as elevated anticardiolipin antibodies, lupus anticoagulant, or a history compatible with antiphospholipid syndrome. It was possible to measure a urinary metabolite of thromboxane A2 in 2 of the patients as an indicator of in vivo platelet activation, and this was markedly elevated in both. In addition, the patients had evidence of ongoing inflammation as indicated by elevated erythrocyte sedimentation rate, hypocomplementemia, and/or elevated levels of anti-DNA antibodies. The findings in these 4 patients suggest that COX-2 inhibitor-treated patients with diseases that predispose to thrombosis should be monitored carefully for this complication.