[show abstract][hide abstract] ABSTRACT: Systemic lupus erythematosus (SLE) is an autoimmune disease with known genetic, epigenetic, and environmental risk factors. To assess the role of DNA methylation in SLE, we collected CD4+ T-cells, CD19+ B-cells, and CD14+ monocytes from 49 SLE patients and 58 controls, and performed genome-wide DNA methylation analysis with Illumina Methylation450 microarrays. We identified 166 CpGs in B-cells, 97 CpGs in monocytes, and 1,033 CpGs in T-cells with highly significant changes in DNA methylation levels (p<1×10(-8)) among SLE patients. Common to all three cell-types were widespread and severe hypomethylation events near genes involved in interferon signaling (type I). These interferon-related changes were apparent in patients collected during active and quiescent stages of the disease, suggesting that epigenetically-mediated hypersensitivity to interferon persists beyond acute stages of the disease and is independent of circulating interferon levels. This interferon hypersensitivity was apparent in memory, naïve and regulatory T-cells, suggesting that this epigenetic state in lupus patients is established in progenitor cell populations. We also identified a widespread, but lower amplitude shift in methylation in CD4+ T-cells (>16,000 CpGs at FDR<1%) near genes involved in cell division and MAPK signaling. These cell type-specific effects are consistent with disease-specific changes in the composition of the CD4+ population and suggest that shifts in the proportion of CD4+ subtypes can be monitored at CpGs with subtype-specific DNA methylation patterns.
[show abstract][hide abstract] ABSTRACT: To reliably transport blood samples for cryoglobulin analysis, we have created a sample transport device containing a mixture of two waxes that solidifies at 38°C and maintains sample temperature at 38°C. Samples arriving at the laboratory at 37 to 38°C increased to 95% from 34% with the use of the device.
[show abstract][hide abstract] ABSTRACT: In patients with systemic lupus erythematosus (SLE), evidence suggests that most vaccines (except live-virus vaccines) are safe, although antibody response may be reduced. This substudy from the phase III, randomized, double-blind, placebo-controlled BLISS-76 trial evaluated the effects of belimumab on preexisting antibody levels against pneumococcal, tetanus, and influenza antigens in patients with SLE.
In BLISS-76, patients with autoantibody-positive, active SLE were treated with placebo or belimumab 1 or 10 mg/kg every 2 weeks for 28 days and every 28 days thereafter, plus standard SLE therapy, for 76 weeks. This analysis included a subset of patients who had received pneumococcal or tetanus vaccine within 5 years or influenza vaccine within 1 year of study participation. Antibodies to vaccine antigens were tested at baseline and Week 52, and percentage changes in antibody levels from baseline and proportions of patients maintaining levels at Week 52 were assessed. Antibody titers were also assessed in a small number of patients vaccinated during the study.
Consistent with preservation of the memory B cell compartment with belimumab treatment, the proportions of patients maintaining antibody responses to pneumococcal, tetanus, and influenza antigens were not reduced. In a small group receiving influenza vaccine on study, antibody responses were frequently lower with belimumab, although titer levels were > 1:10 in all patients treated with 10 mg/kg and in the majority treated with 1 mg/kg.
Treatment with belimumab did not affect the ability of patients with SLE to maintain antibody titers to previous pneumococcal, tetanus, and influenza immunizations. [ClinicalTrials.gov registration number NCT 00410384].
The Journal of Rheumatology 06/2012; 39(8):1632-40. · 3.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: To assess the effects of the B lymphocyte stimulator (BLyS)-specific inhibitor belimumab on immunologic biomarkers, including B cell and T cell populations, and maintenance of antibody titers to prior vaccines in autoantibody-positive systemic lupus erythematosus (SLE) patients.
Pooled data from 2 phase III trials, the Study of Belimumab in Subjects with SLE 52-week (BLISS-52) and 76-week (BLISS-76) trials, comparing belimumab 1 mg/kg or 10 mg/kg versus placebo (plus standard SLE therapy for each group) were analyzed for changes in autoantibody, immunoglobulin, and complement levels. BLISS-76 patients were also analyzed for changes in B cell and T cell populations and effects on prior vaccine-induced antibody levels.
Belimumab-treated patients experienced significant sustained reductions in IgG and autoantibodies and improvement in C3/C4 levels, resulting in greater positive-to-negative conversion rates for IgG anti-double-stranded DNA (anti-dsDNA), anti-Sm, anticardiolipin, and anti-ribosomal P autoantibodies and normalization of hypergammaglobulinemia and low C3/C4 levels. Belimumab-treated patients experienced significant decreases in the numbers of naive and activated B cells, as well as plasma cells, whereas memory B cells and T cell populations did not decrease. Belimumab did not substantially affect preexisting antipneumococcal or anti-tetanus toxoid antibody levels. Post hoc analysis showed greater reductions in SLE disease activity and the risk of severe flares in patients treated with belimumab 10 mg/kg (P≤0.01) who were anti-dsDNA positive and had low C3/C4 levels at baseline. Normalization of the C3 or anti-dsDNA level by 8 weeks, irrespective of therapy, was predictive of a reduced risk of severe flare over 52 weeks.
Belimumab appears to promote normalization of serologic activity and reduce BLyS-dependent B cell subsets in serologically and clinically active SLE. Greater serologic activity may predict a better treatment response to belimumab.
[show abstract][hide abstract] ABSTRACT: To assess the efficacy/safety of the B lymphocyte stimulator inhibitor belimumab plus standard therapy compared with placebo plus standard therapy in active systemic lupus erythematosus (SLE).
In a phase III, multicenter, randomized, placebo-controlled trial, 819 antinuclear antibody-positive or anti-double-stranded DNA-positive SLE patients with scores ≥6 on the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) version of the SLE Disease Activity Index (SLEDAI) were randomized in a 1:1:1 ratio to receive 1 mg/kg belimumab, 10 mg/kg belimumab, or placebo intravenously on days 0, 14, and 28 and then every 28 days for 72 weeks. The primary efficacy end point was the SLE Responder Index (SRI) response rate at week 52 (an SRI response was defined as a ≥4-point reduction in SELENA-SLEDAI score, no new British Isles Lupus Assessment Group [BILAG] A organ domain score and no more than 1 new BILAG B score, and no worsening in physician's global assessment score versus baseline).
Belimumab at 10 mg/kg plus standard therapy met the primary efficacy end point, generating a significantly greater SRI response at week 52 compared with placebo (43.2% versus 33.5%; P = 0.017). The rate with 1 mg/kg belimumab was 40.6% (P = 0.089). Response rates at week 76 were 32.4%, 39.1%, and 38.5% with placebo, 1 mg/kg belimumab, and 10 mg/kg belimumab, respectively. In post hoc sensitivity analyses evaluating higher SELENA-SLEDAI score thresholds, 10 mg/kg belimumab achieved better discrimination at weeks 52 and 76. Risk of severe flares over 76 weeks (based on the modified SLE Flare Index) was reduced with 1 mg/kg belimumab (34%) (P = 0.023) and 10 mg/kg belimumab (23%) (P = 0.13). Serious and severe adverse events, including infections, laboratory abnormalities, malignancies, and deaths, were comparable across groups.
Belimumab plus standard therapy significantly improved SRI response rate, reduced SLE disease activity and severe flares, and was generally well tolerated in SLE.
[show abstract][hide abstract] ABSTRACT: Death Receptor 5 (DR5) induces apoptosis in various types of cells and is a potential therapeutic target. We have investigated whether targeting DR5 could be used to eliminate pathogenic B lymphocytes from systemic lupus erythematosus (SLE) patients. We examined DR5 expression and function on B lymphocytes from healthy controls subjects, SLE patients, and human tonsil. DR5 was expressed similarly on all B cell subpopulations, including resting and activated B cells. Expression of DR5 was equivalent on B cells from SLE patients and healthy subjects. Additionally, DR5 expression was unchanged after B lymphocyte stimulation. However, B cells were resistant to DR5-induced apoptosis, including after in vitro activation. No changes in subsets of B cells were observed in subjects of a trial of CS-1008, an agonist anti-DR5. While DR5 shows promise as a way to selectively eliminate tumor cells and activated synoviocytes, these data suggest DR5 alone cannot be used as a target to remove pathogenic SLE B cells.
[show abstract][hide abstract] ABSTRACT: 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).
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.
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), physician's 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.
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.
[show abstract][hide abstract] ABSTRACT: To describe a new systemic lupus erythematosus (SLE) responder index (SRI) based on a belimumab phase II SLE trial and demonstrate its potential utility in SLE clinical trials.
Data from a randomized, double-blind, placebo-controlled study in 449 patients of 3 doses of belimumab (1, 4, 10 mg/kg) or placebo plus standard of care therapy (SOC) over a 56-week period were analyzed. The Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and British Isles Lupus Assessment Group (BILAG) SLE disease activity instruments, the Short Form 36 health survey, and biomarker analyses were used to create a novel SRI. Response to treatment in a subset of 321 serologically active SLE patients (antinuclear antibodies >/=1:80 and/or anti-double-stranded DNA antibodies >/=30 IU/ml) at baseline was retrospectively evaluated using the SRI.
SRI response is defined as 1) a >/=4-point reduction in SELENA-SLEDAI score, 2) no new BILAG A or no more than 1 new BILAG B domain score, and 3) no deterioration from baseline in the physician's global assessment by >/=0.3 points. In serologically active patients, the addition of belimumab to SOC resulted in a response in 46% of patients at week 52 compared with 29% of the placebo patients (P = 0.006). SRI responses were independent of baseline autoantibody subtype.
This evidence-based evaluation of a large randomized, placebo-controlled trial in SLE resulted in the ability to define a robust responder index based on improvement in disease activity without worsening the overall condition or the development of significant disease activity in new organ systems.
[show abstract][hide abstract] ABSTRACT: IgA nephropathy (IgAN) is characterized by circulating immune complexes composed of galactose-deficient IgA1 and a glycan-specific IgG antibody. These immune complexes deposit in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgAN. To define the precise specificities and molecular properties of the IgG antibodies, we generated EBV-immortalized IgG-secreting lymphocytes from patients with IgAN and found that the secreted IgG formed complexes with galactose-deficient IgA1 in a glycan-dependent manner. We cloned and sequenced the heavy- and light-chain antigen-binding domains of IgG specific for galactose-deficient IgA1 and identified an A to S substitution in the complementarity-determining region 3 of the variable region of the gene encoding the IgG heavy chain in IgAN patients. Furthermore, site-directed mutagenesis that reverted the residue to alanine reduced the binding of recombinant IgG to galactose-deficient IgA1. Finally, we developed a dot-blot assay for the glycan-specific IgG antibody that differentiated patients with IgAN from healthy and disease controls with 88% specificity and 95% sensitivity and found that elevated levels of this antibody in the sera of patients with IgAN correlated with proteinuria. Collectively, these findings indicate that glycan-specific antibodies are associated with the development of IgAN and may represent a disease-specific marker and potential therapeutic target.
The Journal of clinical investigation 07/2009; 119(6):1668-77. · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: This 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).
Seventy 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. chi2 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.
The 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.
Belimumab was well tolerated and reduced peripheral B-cell levels in SLE patients. These data support further studies of belimumab in autoimmune disorders.
Arthritis research & therapy 10/2008; 10(5):R109. · 4.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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).
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.
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).
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.
[show abstract][hide abstract] ABSTRACT: Guidelines and recommendations developed and/or endorsed by the American College of Rheumatology (ACR) are intended to provide guidance for particular patterns of practice and not to dictate the care of a particular patient. The ACR considers adherence to these guidelines and recommendations to be voluntary, with the ultimate determination regarding their application to be made by the physician in light of each patient's individual circumstances. Guidelines and recommendations are intended to promote beneficial or desirable outcomes but cannot guarantee any specific outcome. Guidelines and recommendations developed or endorsed by the ACR are subject to periodic revision as warranted by the evolution of medical knowledge, technology, and practice.
[show abstract][hide abstract] ABSTRACT: To determine whether receptors for B lymphocyte stimulator (BLyS) are altered on B cells of patients with systemic lupus erythematosus (SLE).
Total available receptors for BLyS were measured by analysis of binding of recombinant soluble BLyS to peripheral blood B cells in 36 SLE patients, 29 healthy controls, and 10 disease controls. Antibodies to the receptors BAFF-R, BCMA, and TACI were used to define expression of the individual BLyS receptors on subsets of B cells in blood, spleen, and tonsils. Two different antibodies to BAFF-R, which were differentially sensitive to BAFF-R occupancy, were used to compare BAFF-R on B cells in an additional 20 healthy subjects and 25 SLE patients. Assays of B cell survival after stimulation in vitro were used to determine the sensitivity of B cells to exogenous BLyS.
Total available receptors for BLyS were decreased in patients with SLE, independent of changes of subsets in the blood in these patients. The decrease correlated with changes in disease activity. Although total surface BAFF-R was not significantly different between healthy controls and SLE patients, BAFF-R was occupied in SLE patients. B cells from these patients were less responsive to exogenous BLyS.
BAFF-R is consistently occupied on blood B cells in SLE. Occupancy of BAFF-R on blood B cells is likely to contribute to disease mechanisms in SLE and could serve as a biomarker of disease activity. Targeting BLyS as a therapeutic strategy will require overcoming the persistent binding of BLyS to BAFF-R.
[show abstract][hide abstract] ABSTRACT: Objective
To evaluate the effectiveness of a personal digital assistant (PDA)–based clinical decision support system (CDSS) on nonsteroidal anti-inflammatory drug (NSAID) prescribing safety in the outpatient setting.DesignThe design was a randomized, controlled trial conducted in a university-based resident clinic. Internal medicine residents received a PDA-based CDSS suite. For intervention residents, the CDSS included a prediction rule for NSAID-related gastrointestinal risk assessment and treatment recommendations. Unannounced standardized patients (SPs) trained to portray musculoskeletal symptoms presented to study physicians. Safety outcomes were assessed from the prescriptions given to the SPs. Each prescription was reviewed by a committee of clinicians blinded to participant, intervention group assignment, and baseline or follow-up status.MeasurementsPrescriptions were judged as safe or unsafe. The main outcome measure was the differential change in unsafe prescribing of NSAIDs for the intervention versus the control group.ResultsAt baseline, the mean proportion of cases per physician with unsafe prescriptions for the two groups was similar (0.27 vs. 0.29, p > 0.05). Controlling for baseline performance, intervention participants prescribed more safely than controls after receiving the CDSS (0.23 vs. 0.45 [F = 4.24, p < 0.05]). With the CDSS, intervention participants documented more complete assessment of patient gastrointestinal risk from NSAIDs.Conclusion
Participants provided with a PDA-based CDSS for NSAID prescribing made fewer unsafe treatment decisions than participants without the CDSS.
[show abstract][hide abstract] ABSTRACT: Objective. To determine if preexposure of human articular cartilage to activated neutrophils alters rheumatoid synovial fibroblast adhesion to human articular cartilage.Methods. Human articular cartilage was exposed to either activated neutrophils, interleukin-1, or supernatants obtained from activated neutrophils that had been treated with different protease inhibitors. Radiolabeled rheumatoid synovial fibroblasts were then incubated with the cartilage and the number of counts associated with the cartilage was determined.Results. Pretreatment of human articular cartilage with either activated neutrophils or supernatants obtained from activated neutrophils enhanced subsequent rheumatoid synovial fibroblast adhesion. In contrast, interleukin-1 treatment of cartilage did not alter the adhesion of synovial fibroblasts. The enhanced adhesion could be attenuated by pretreatment of the neutrophil supernatants with either diisopropylfluoro-phosphonate or EGTA and almost completely abolished by using both inhibitors.Conclusion. This study demonstrates that adhesion of rheumatoid synovial fibroblasts to human articular cartilage can be enhanced by exposing the cartilage to proteases released by neutrophils. These results suggest that neutrophil products may play a role in enhancing adhesion of rheumatoid synovium to cartilage in vivo.
[show abstract][hide abstract] ABSTRACT: Antineutrophil cytoplasmic Abs (ANCA) can activate neutrophils in an FcgammaR-dependent manner, but the link between this ANCA-induced effect and mononuclear cell activation with the characteristic granuloma formation of Wegener's granulomatosis is unclear. Human alpha-defensins, small cationic antimicrobial peptides, are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and monocytes. In this study, we quantitated the release of alpha-defensins (human neutrophil peptides 1-3) from human neutrophils after targeted FcgammaR cross-linking (XL). Homotypic XL of FcgammaRIIa, FcgammaRIIIb, or heterotypic XL of both receptors resulted in significant release of alpha-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-proteinase 3). This release of alpha-defensins, as well as of other granule constituents (ANCA targets anti-proteinase 3 and myeloperoxidase and elastase), was significantly greater in donors homozygous for the NA1 allele of FcgammaRIIIb than in donors homozygous for NA2. Interestingly, the ANCA-induced release was completely inhibited by the IgG Fc-binding peptide TG19320, which blocks the IgG-Fc region from binding to FcgammaR. Based on their chemotactic properties, alpha-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma formation. The greater activity of the FcgammaRIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele.
The Journal of Immunology 01/2004; 171(11):6090-6. · 5.52 Impact Factor