Objective. To evaluate the efficacy and safety of rituximab in a randomized, double-blind, placebocontrolled phase III trial in patients with lupus nephritis treated concomitantly with mycophenolate mofetil (MMF) and corticosteroids.Methods. Patients (n ‫؍‬ 144) with class III or class IV lupus nephritis were randomized 1:1 to receive rituximab (1,000 mg) or placebo on days 1, 15, 168, and 182. The primary end point was renal response status at week 52.Results. Rituximab depleted peripheral CD19؉ B cells in 71 of 72 patients. The overall (complete and partial) renal response rates were 45.8% among the 72 patients receiving placebo and 56.9% among the 72 patients receiving rituximab (P ‫؍‬ 0.18); partial responses accounted for most of the difference. The primary end point (superior response rate with rituximab) was not achieved. Eight placebo-treated patients and no rituximab-treated patients required cyclophosphamide rescue therapy through week 52. Statistically significant improvements in serum complement C3, C4, and antidouble-stranded DNA (anti-dsDNA) levels were observed among patients treated with rituximab. In both treatment groups, a reduction in anti-dsDNA levels greater than the median reduction was associated with reduced proteinuria. The rates of serious adverse events, including infections, were similar in both groups. Neutropenia, leukopenia, and hypotension occurred more frequently in the rituximab group.Conclusion. Although rituximab therapy led to more responders and greater reductions in anti-dsDNA and C3/C4 levels, it did not improve clinical outcomes after 1 year of treatment. The combination of rituximab with MMF and corticosteroids did not result in any new or unexpected safety signals.Lupus nephritis (LN) may be observed in up to 50% of patients with systemic lupus erythematosus (SLE) and is associated with a poor prognosis (1). Although renal response rates among patients receiving standard treatment of proliferative LN may approach ClinicalTrials.gov identifier: NCT00282347.

Objective. B lymphocyte depletion has recently emerged as a promising approach to the treatment of systemic lupus erythematosus (SLE). As part of a phase I/II dose-ranging trial of rituximab in the treatment of SLE, we evaluated the fate of discrete B cell subsets in the setting of selective depletion by anti-CD20 monoclonal antibody and during the B cell recovery phase.Methods. B cell depletion and phenotype were examined by flow cytometry of peripheral blood mononuclear cells for CD19, CD20, CD27, IgD, and CD38 expression. Changes in autoreactive B lymphocytes and plasma cells were assessed by determination of serum autoantibody levels (anti-double-stranded DNA and VH4.34) and by direct monitoring of a unique autoreactive B cell population bearing surface antibodies whose heavy chain is encoded by the VH4.34 gene segment.Results. Compared with normal controls, SLE patients displayed several abnormalities in peripheral B cell homeostasis at baseline, including naive lymphopenia, expansion of a CD27؊,IgD؊ (double negative) population, and expansion of circulating plasmablasts. Remarkably, these abnormalities resolved after effective B cell depletion with rituximab and immune reconstitution. The frequency of autoreactive VH4.34 memory B cells also decreased 1 year posttreatment, despite the presence of low levels of residual memory B cells at the point of maximal B cell depletion and persistently elevated serum autoantibody titers in most patients.Conclusion. This study is the first to show evidence that in SLE, specific B cell depletion therapy with rituximab dramatically improves abnormalities in B cell homeostasis and tolerance that are characteristic of this disease. The persistence of elevated autoantibody titers may reflect the presence of low levels of residual autoreactive memory B cells and/or long-lived autoreactive plasma cells.

Objective. Despite wide use of the anti-CD20 monoclonal antibody rituximab in the treatment of B cell lymphomas, the mechanism by which it causes B cell depletion remains a subject of controversy. As part of an ongoing phase I/II trial of rituximab in the treatment of systemic lupus erythematosus (SLE), we sought to determine whether the effectiveness of B cell depletion was influenced by polymorphisms of Fc receptors (FcR) on effector cells.Methods. During rituximab treatment of 12 SLE patients, B cell depletion was monitored as a function of the serum rituximab level and Fc␥RIIa and Fc␥RIIIa genotypes at baseline and at 1 month and 2 months after treatment. FcR genotypes were determined by polymerase chain reaction. Serum levels of rituximab were measured by enzyme-linked immunosorbent assay (ELISA). B lymphocyte percentages were assessed by flow cytometry.Results. B cell depletion was highly variable in this patient cohort, with B cell percentages at the 1-2-month posttreatment nadir ranging from undetectable (<0.1 cell/ l) to 16% (ϳ30 cells/ l) of the total peripheral blood lymphocytes. At 2 months posttreatment, B cell percentages were highly correlated with both the serum rituximab level and the Fc␥RIIIa genotype (R 2 ‫؍‬ 0.75, P ‫؍‬ 0.002). The Fc␥RIIIa genotype was a significant independent predictor of the efficacy of B cell depletion (P ‫؍‬ 0.019). Conclusion. These results highlight the potential variability of B cell depletion by rituximab in the treatment of autoimmune disease and indicate that Fc receptors are an important determinant of that variability. The findings further suggest the importance of antibody-dependent cell-mediated cytotoxicity and/or apoptosis induction via Fc␥RIIIa-expressing effector cells in the mechanism of B cell depletion by this widely used monoclonal antibody.Rituximab is a chimeric mouse-human monoclonal antibody against the B cell-specific antigen CD20, a cell surface protein believed to function in B cell cycle initiation and differentiation (1-3). CD20 is first expressed in the early pre-B cell stage, and it remains present until terminal differentiation into plasma cells. The CD20 antigen represents an ideal target for immunotherapy of B cell lymphomas and, more recently, B cell-mediated autoimmune diseases due to its high and relatively sustained expression on malignant and normal B cells (4). Rituximab can effectively deplete B cells for several months and, as such, represents an effective treatment of B cell lymphoma (5).Several potential mechanisms have been proposed as mediators of the effects of rituximab (6). Thus, in vitro studies have demonstrated that rituximab bound to CD20ϩ cells induces complement-dependent cytotoxicity (CDC) and, in the presence of effector cells, antibody-dependent cell-mediated cytotoxicity (ADCC) (5). Additionally, rituximab can induce B cell apoptosis when crosslinked by Fc␥ receptor (Fc␥R)-bearing cells (7). The relative importance of antibody-mediated apoptosis, CDC, and ADCC in the depletion of B cells by rituximab remains to be...

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Objective. To investigate the mechanisms whereby tumor necrosis factor ␣ (TNF␣) increases osteoclastogenesis in vivo.Methods. TNF␣-transgenic (TNF-Tg) and wildtype mice injected with TNF␣ were studied. In vitro osteoclastogenesis assays, monocyte colony-forming assays, and fluorescence-activated cell sorting were performed using splenocytes, peripheral blood mononuclear cells (PBMCs), and bone marrow cells to quantify and characterize osteoclast precursors (OCPs). Etanercept, a TNF␣ antagonist, was used to block TNF␣ activity in vivo. The effects of TNF␣ on proliferation, apoptosis, and differentiation of OCPs were assessed using 5-bromo-2-deoxyuridine labeling, annexin V staining, and reverse transcriptase-polymerase chain reaction.Results Conclusion. Systemic TNF␣ induces a marked increase in circulating OCPs that is reversible by anti-TNF therapy and may result from their mobilization from bone marrow. Our findings provide a new mechanism whereby TNF␣ stimulates osteoclastogenesis in patients with inflammatory arthritis, suggesting that CD11b؉ PBMCs could be used to evaluate a patient's potential for erosive disease and the efficacy of anti-TNF therapy.