Kevin P. Baker

Cutting Edge: A Role for B Lymphocyte Stimulator in Systemic Lupus Erythematosus

Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member TACI Is a High Affinity Receptor for TNF Family Members APRIL and BLyS

An expression cloning approach was employed to identify the receptor for B-lymphocyte stimulator (BLyS) and identified the tumor necrosis factor receptor superfamily member TACI as a BLyS-binding protein. Expression of TACI in HEK293T cells confers on the cells the ability to bind BLyS with subnanomolar affinity. Furthermore, a TACI-Fc fusion protein recognizes both the cleaved, soluble form of BLyS as well as the membrane BLyS present on the cell surface of a recombinant cell line. TACI mRNA is found predominantly in B-cells and correlates with BLyS binding in a panel of B-cell lines. We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-κB responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.

BLyS and APRIL Form Biologically Active Heterotrimers That Are Expressed in Patients with Systemic Immune-Based Rheumatic Diseases

BLyS and APRIL are two members of the TNF superfamily that are secreted by activated myeloid cells and have costimulatory activity on B cells. BLyS and APRIL share two receptors, TACI and BCMA, whereas a third receptor, BAFF-R, specifically binds BLyS. Both BLyS and APRIL have been described as homotrimeric molecules, a feature common to members of the TNF superfamily. In this study, we show that APRIL and BLyS can form active heterotrimeric molecules when coexpressed and that circulating heterotrimers are present in serum samples from patients with systemic immune-based rheumatic diseases. These findings raise the possibility that active BLyS/APRIL heterotrimers may play a role in rheumatic and other autoimmune diseases and that other members of the TNF ligand superfamily may also form active soluble heterotrimers.

Inverse association between circulating APRIL levels and serological and clinical disease activity in patients with systemic lupus erythematosus

Objective: To assess longitudinal expression of a proliferation-inducing ligand (APRIL) in patients with systemic lupus erythematosus (SLE) and its correlation with B lymphocyte stimulator (BLyS) expression, serum anti-dsDNA titres, and clinical disease activity. Methods: Sixty eight patients with SLE were longitudinally followed up for a median of 369 days. At each visit the physician assessed disease activity by SLEDAI, and blood was collected for determination of serum APRIL and BLyS levels and of blood APRIL and BLyS mRNA levels. Fifteen normal control subjects underwent similar laboratory evaluation. Results: Dysregulation of APRIL was not as great as that of BLyS. Changes in serum levels of APRIL and BLyS over time were usually discordant, whereas blood levels of APRIL and BLyS mRNA strongly paralleled each other. Serum APRIL levels modestly, but significantly, inversely correlated with serum anti-dsDNA titres in anti-dsDNA positive patients analysed in aggregate. Moreover, serum APRIL levels modestly, but significantly, inversely correlated with clinical disease activity in all patients analysed in aggregate. Conclusion: Serum levels of APRIL and BLyS are differentially regulated. APRIL may serve as a down modulator of serological and/or clinical autoimmunity in patients with SLE. This may have important ramifications for BLyS targeted treatment, and it remains to be determined whether agents which neutralise only BLyS will be preferable to agents which neutralise both BLyS and APRIL.

AlbugraninTM, a Recombinant Human Granulocyte Colony Stimulating Factor (G-CSF) Genetically Fused to Recombinant Human Albumin Induces Prolonged Myelopoietic Effects in Mice and Monkeys

AbstractPurpose. AlbugraninTM fusion protein is recombinant granulocyte colony stimulating factor (rG-CSF) genetically fused at its N-terminus to the C-terminus of recombinant serum human albumin and is expected to have a relatively long half-life compared with rG-CSF alone. In this study, the pharmacodynamics and pharmacokinetics of Albugranin were evaluated in BDF1 mice and cynomolgus monkeys. Methods. Single doses of Albugranin (0.25-5 mg/kg) or Filgrastim (methionyl rG-CSF, 0.25, or 1.25 mg/kg) were administered subcutaneously (SC) to mice and multiple doses of Albugranin (25-100 μg/kg every 4 or 7 days) or Filgrastim (5 μg/kg daily) were administered SC for 14 days to monkeys for hematologic evaluation. For pharmacokinetics studies, mice were injected intravenously (IV) or SC with single doses of Albugranin (0.25-1.25 mg/kg) or Filgrastim (0.25 mg/kg) and monkeys were injected SC with multiple doses of Albugranin (100-1000 μg/kg once weekly for 5 weeks). Plasma levels of Albugranin and Filgrastim were measured by enzyme-linked immunosorbent assay. Results. In mice, administration of Albugranin effectively increased the number of peripheral granulocytes and mobilized hematopoietic progenitor cells for up to 5 days. The magnitude and duration of this effect were dose-dependent. In contrast, administration of Filgrastim resulted in a small increase in both cell types on day 1 only. Albugranin administered to cynomolgus monkeys caused an increase in peripheral neutrophils, with a less prominent increase in peripheral monocytes. Albugranin-induced neutrophilia peaked 24 h following each dose administration. Administration of Filgrastim daily in monkeys resulted in moderate increases in neutrophils that were maximal on days 8-12 during the course of treatment. Compared with Filgrastim, Albugranin had a longer terminal half-life (t1/2,term), and mean residence time (MRT), and slower clearance (CL/F) in mice. The t1/2,term, MRT, and CL/F of Albugranin following SC administration to BDF1 mice were 5.6-5.7 h, 16.7-20.7 h, and 6.37-12.2 mL/h/kg, respectively, compared with 2.54 h, 4.9 h, and 164 mL/h/kg, respectively for Filgrastim. In cynomolgus monkeys, the corresponding values of t1/2,term, MRT, and CL/F for Albugranin were 7.73-13.3 h, 19.4-27.3 h, and 7.90-27.5 mL/h/kg, respectively, for doses of 100-1000 μg/kg. An exposure-response relationship that could be empirically described with a simple Emax model with baseline was found between day 15 absolute neutrophil count and area under the curve following the first dose in cynomolgus monkeys. Conclusion. The sustained activity of Albugranin in mice and monkeys demonstrated in these studies suggests that this agent could be given less frequently than Filgrastim to achieve similar therapeutic effects in patients.