Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40

SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions

The Use of a Stringent Selection System Allows the Identification of DNA Elements that Augment Gene Expression

The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner

The Cyclic AMP Cascade Is Altered in the Fragile X Nervous System

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3′, 5′-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling

Endothelial TRPV4 channels modulate vascular tone by Ca2+‐induced Ca2+ release at inositol 1,4,5‐trisphosphate receptors

Background and Purpose: The TRPV4 ion channels are Ca2+ permeable, non‐selective cation channels that mediate large, but highly localized, Ca2+ signals in the endothelium. The mechanisms that permit highly localized Ca2+ changes to evoke cell‐wide activity are incompletely understood. Here, we tested the hypothesis that TRPV4‐mediated Ca2+ influx activates Ca2+ release from internal Ca2+ stores to generate widespread effects. Experimental Approach: Ca2+ signals in large numbers (~100) of endothelial cells in intact arteries were imaged and analysed separately. Key Results: Responses to the TRPV4 channel agonist GSK1016790A were heterogeneous across the endothelium. In activated cells, Ca2+ responses comprised localized Ca2+ changes leading to slow, persistent, global increases in Ca2+ followed by large propagating Ca2+ waves that moved within and between cells. To examine the mechanisms underlying each component, we developed methods to separate slow persistent Ca2+ rise from the propagating Ca2+ waves in each cell. TRPV4‐mediated Ca2+ entry was required for the slow persistent global rise and propagating Ca2+ signals. The propagating waves were inhibited by depleting internal Ca2+ stores, inhibiting PLC or blocking IP3 receptors. Ca2+ release from stores was tightly controlled by TRPV4‐mediated Ca2+ influx and ceased when influx was terminated. Furthermore, Ca2+ release from internal stores was essential for TRPV4‐mediated control of vascular tone. Conclusions and Implications: Ca2+ influx via TRPV4 channels is amplified by Ca2+‐induced Ca2+ release acting at IP3 receptors to generate propagating Ca2+ waves and provide a large‐scale endothelial communication system. TRPV4‐mediated control of vascular tone requires Ca2+ release from the internal store