Identification of a potent anti-IL-15 antibody with opposing mechanisms of action in vitro and in vivo.

BACKGROUND AND PURPOSE: Interleukin-15 (IL-15) is important in the activation and proliferation of lymphocytic cell populations and is implicated in inflammatory disease. We report the characterization of a novel monoclonal antibody DISC0280 which is specific for human IL-15. EXPERIMENTAL APPROACH: DISC0280 was characterized in a direct binding assay of IL-15 with IL-15 receptor α (IL-15Rα) and by its ability to alter IL-15 mediated proliferation of a range of cell lines (cytotoxic T lymphocyte line-2, M-07e, KIT225). A pharmacodynamic model injecting male C57/BL6 mice with IL-15 or IL-15/IL-15Rα, with or without DISC0280, and assessing changes in lymphocytic cell populations and serum cytokines was utilized. KEY RESULTS: DISC0280 inhibited the binding of IL-15 to IL-15Rα and also potently inhibits IL-15 dependent proliferation of cells expressing IL-15Rα, shared interleukin 2/ interleukin 15 receptor ß chain (IL-15Rß) and common gamma chain (γ(c) ). DISC0280 also inhibited the IL-15 dependent proliferation of M-07e cells that only express IL-15Rß/γ(c) subunits. Human IL-15 injected into mice caused an increase in NK1.1(+) and CD3(+) cells in the spleen and peripheral blood and these effects were unexpectedly potentiated by giving DISC0280 with human IL-15. This increase in cells caused by DISC0280/IL-15 co-administration was greater than that observed when IL-15 was administered complexed with soluble IL-15Rα. CONCLUSIONS AND IMPLICATIONS: The ability of DISC0280 to bind to the IL-15Rα-binding site on IL-15 allows trans-presentation of IL-15 by DISC0280 in vivo, similar to the trans-presentation by soluble IL-15Rα. DISC0280 may be therefore suitable as a clinical substitute for IL-15.

Preliminary crystallographic studies of an extremely thermostable KDG aldolase from Sulfolobus solfataricus.

Crystals have been grown of 2-keto-3-deoxygluconate aldolase (KDG aldolase) from the hyperthermophilic archaeon Sulfolobus solfataricus that diffract to 2.2 A resolution. The enzyme catalyses the reversible aldol cleavage of 2-keto-3-dexoygluconate to pyruvate and glyceraldehyde, the third step of a modified non-phosphorylated Entner-Doudoroff pathway of glucose oxidation. S. solfataricus grows optimally at 353 K and the enzyme itself has a half-life of 2.5 h at 373 K. Knowledge of the crystal structure of KDG aldolase will further understanding of the basis of protein hyperthermostability and create a target for site-directed mutagenesis of active-site residues, with the aim of altering substrate specificity. Three crystal forms have been obtained: orthorhombic crystals of space group P2(1)2(1)2(1), which diffract to beyond 2.15 A, monoclinic crystals of space group C2, which diffract to 2.2 A, and cubic crystals of space group P4(2)32, which diffract to 3.4 A.

An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates.

Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 degrees C. It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C(6) to C(3) aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde. Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S. solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme. The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S. solfataricus cells. The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 degrees C of 2.5 h, and is equally active with both d- and l-glyceraldehyde. It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH(4).

"Low-dose" growth hormone therapy during peritoneal dialysis or following renal transplantation.

The minimal effective dose of growth hormone (GH) to promote growth in children on dialysis or following renal transplantation remains unsettled. In order to study the issue, "low-dose" GH was administered to children with end-stage renal disease (ESRD) receiving chronic automated peritoneal dialysis (APD, n = 6, 4 males, 2 females) or following renal transplantation (T, n = 9, 8 males, 1 female). No APD patient was GH deficient, while 1 T patient (no. 2) had data consistent with GH deficiency, although he was obese (body mass index = 34 kg/m2). The mean dose of GH after 6 and 12 months of treatment was 0.16 +/- 0.02 and 0.22 +/- 0.07 versus 0.16 +/- 0.03 and 0.27 +/- 0.21 mg/kg per week for APD and T patients, respectively. When analyzing all patients, there were no significant differences before or after 6 and 12 months of GH therapy within or between the two groups, in terms of height velocity, bone age, renal function (in the T group) and height Z-scores (Z-Ht). However, the height velocity Z-score (Z-HV) increased significantly at 6 and 12 months compared with baseline in the APD patients only (P < 0.05). When the 2 T patients with the most impaired renal function were excluded from the analysis, Z-HV also increased significantly in the T patients after 12 months of GH (P < 0.02). We conclude that following "low-dose" GH therapy, children with ESRD treated with APD or T have similar increases in HV, allowing maintenance of Z-Ht but not "catch-up" growth.