Autoantibodies to variable heavy (VH) chain Ig sequences in humans impact the safety and clinical pharmacology of a VH domain antibody antagonist of TNF-α receptor 1.

PURPOSE: To investigate the impact of a new class of anti-Ig autoantibodies reactive with variable heavy (VH) chain framework sequences (human anti-VH autoantibodies) on the pharmacology and safety of an anti-TNFR1 VH domain antibody (GSK1995057) in healthy human subjects. METHODS: Single-blind, randomised, placebo-controlled dose escalation study in which healthy males (n = 28) received a single GSK1995057 intravenous infusion of 0.0004, 0.002 and 0.01 mg/kg. All enrolled subjects were pre-screened for human anti-VH (HAVH) autoantibody status and prospectively stratified accordingly. Serum samples from drug-naïve, HAVH-positive volunteers were used to investigate the effect of HAVH/GSK1995057 complexes on the activation of TNFR1 and cytokine release in vitro. RESULTS: Human anti-VH autoantibodies were detected in approximately 50 % of drug-naïve healthy human subjects and clinical and in vitro studies were performed to evaluate their impact on the pharmacology and safety of GSK1995057. We demonstrated that formation of HAVH autoantibody/GSK1995057 complexes activated TNFR1 and caused cytokine release in vitro in some, but not all, of the human cell types tested. When GSK1995057 was administered to healthy subjects, clinical and physiological signs of cytokine release were observed in two HAVH autoantibody-positive subjects following GSK1995057 infusion. In vitro, HAVH autoantibody levels correlated with TNFR1-dependent cytokine release and propensity for cytokine release in humans following GSK1995057 dosing. CONCLUSIONS: Our data support a greater focus on the impact of pre-existing, drug-reactive autoantibodies on the development of antibody fragments and biotherapeutics targeting cell surface receptors.

X-linked inhibitor of apoptosis protein functions as a cofactor in transforming growth factor-beta signaling.

X-linked inhibitor of apoptosis protein (XIAP) is a potent suppressor of apoptotic cell death, which functions by directly inhibiting caspases, the principal effectors of apoptosis. Here we report that XIAP can also function as a cofactor in the regulation of gene expression by transforming growth factor-beta (TGF-beta). XIAP, but not the related proteins c-IAP1 or c-IAP2, associated with several members of the type I class of the TGF-beta receptor superfamily and potentiated TGF-beta-induced signaling. Although XIAP-mediated activation of c-Jun N-terminal kinase and nuclear factor kappa B was found to require the TGF-beta signaling intermediate Smad4, the ability of XIAP to suppress apoptosis was found to be Smad4-independent. These data implicate a role for XIAP in TGF-beta-mediated signaling that is distinct from its anti-apoptotic functions.

Transforming growth factor-beta receptor-associated protein 1 is a Smad4 chaperone.

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-beta receptors and suggested to play a role in TGF-beta signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-beta and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-beta signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-beta signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.

A cellular automaton model of cellular signal transduction.

On the basis of cellular automata models, a software specifically tailored to model biochemical reactions involved in cellular signal transduction was implemented on a personal computer. Recent data regarding desensitization processes in mouse Leydig cells are used to simulate the underlying reactions of signal transduction. Pretreatment of real Leydig cells with different molecules results in a modification of the signal transduction cascade leading to a diminished response of the cells during subsequent stimulations. The model is capable of simulating the complex behavior of this intracellular second messenger production in a qualitative and semi-quantitative way. The results indicate that quantitative simulations on a molecular level will be possible once appropriate computer hardware is available. The simulations and results of the cellular automaton presented are easily described and comprehended, which make it a useful tool that will facilitate research in cellular signal transduction and other fields covering complex reaction networks.

Leukostasis followed by hemorrhage complicating the initiation of chemotherapy in patients with acute myeloid leukemia and hyperleukocytosis: a clinicopathologic report of four cases.

BACKGROUND: Pulmonary and cerebral leukostasis, or parenchymal hemorrhage in these organs, are well-known early complications developing in patients with acute myeloid leukemia (AML), particularly when myelomonocytic features, hyperleukocytosis, and/or a coagulation disorder are initially present. Commonly, these complications arise during increasing leukocyte counts (WBCs). METHODS: The authors describe four patients with AML and hyperleukocytosis who developed leukostasis followed by parenchymal hemorrhage. RESULTS: Bleeding in all patients occurred while their WBCs were decreasing following cytosine-arabinoside chemotherapy, and in the absence of disseminated intravascular coagulation or severe thrombocytopenia. Radiologic and histopathologic findings underscoring possible mechanisms are presented in the article. CONCLUSIONS: Alterations of cell adhesion associated with chemotherapy-induced blast lysis or cellular differentiation are possible factors contributing to this particular sequence (cytosine arabinoside-based chemotherapy, leukostasis, and subsequent hemorrhage). Prophylactic measures for managing this early complication of AML treatment include leukapheresis to reduce the WBC prior to the initiation of chemotherapy.

LH/hCG-receptor is coupled to both adenylate cyclase and protein kinase C signaling pathways in isolated mouse Leydig cells.

The aim of this study was to examine whether or not a protein kinase C-dependent pathway is involved in the desensitization process of the LH/hCG-receptor-linked adenylate cyclase system in isolated mouse Leydig cells. Treatment of these cells with the phorbol ester, 4-ß-phorbol 12-myristate 13-acetate (PMA) leads to a translocation (and a putative activation) of protein kinase C from the cytosol to the plasma membrane, as evidenced by the Western blotting procedure using particulate and cytosolic fractions of Percoll-purified mouse Leydig cells. A similar translocation is also observed following the treatment of mouse Leydig cells with hCG. Data obtained show that this effect is time-dependent and is mediated specifically through the LH/hCG-receptor. Furthermore, we show that the treatment of Leydig cells with either PMA or hCG leads to a desensitization of the adenylate cyclase stimulated with hCG, hCG plus GppNHp or AIF (4) (-) . This desensitization was not accompanied by a change in the [(125)I]-hCG binding to membrane receptors. Thus we provide here direct evidence that hCG is capable of activating protein kinase C. In addition, we postulate that PMA as well as hCG-treatment leads to a lesion located at a site distal to the receptor/G-protein interaction but proximal to the adenylate cyclase activation and that the translocation (and activation) of protein kinase C may be a common mechanism involved in this desensitizing effect caused by both PMA and hCG on Leydig cells.