We have generated mice carrying a homozygous null mutation in the granulocyte colony-stimulating factor receptor (G-CSFR) gene. G-CSFR-deficient mice have decreased numbers of phenotypically normal circulating neutrophils. Hematopoietic progenitors are decreased in the bone marrow, and the expansion and terminal differentiation of these progenitors into granulocytes is impaired. Neutrophils isolated from G-CSFR-deficient mice have an increased susceptibility to apoptosis, suggesting that the G-CSFR may also regulate neutrophil survival. These data confirm a role for the G-CSFR as a major regulator of granulopoiesis in vivo and provide evidence that the G-CSFR may regulate granulopoiesis by several mechanisms. However, the data also suggest that G-CSFR-independent mechanisms of granulopoiesis must exist.

We investigated the functions of Akt during vascular development and remodeling by using an inducible endothelial cell-specific driver of the dominant-active myrAkt. We found that sustained signaling in response to overexpression of myrAkt led to embryonic lethality, edema, and vascular malformations. In addition to the morphological malformations, the vascular phenotype was consistent with a failure in remodeling, such that the normal patterning and vessel hierarchy was disturbed. Examination of the well studied retinal vasculature during the remodeling phases revealed that transient expression of myrAkt was capable of altering the normal response to oxygen-induced remodeling without causing vascular malformations. These findings suggest that physiological levels of Akt signaling modulated microvascular remodeling and support the hypothesis that, although Akt may be required for vascular growth and homeostasis, appropriate downregulation is also an essential aspect of normal vascular patterning.Akt͞PKB ͉ angiogenesis ͉ retina E ndothelial cell survival and apoptosis have been studied extensively with respect to micro-and macrovascular disease in diabetes, cardiovascular disease, sepsis, and after transplant surgery (1). As a desired effect of therapy, endothelial cell apoptosis has been explored in response to antiangiogenic treatments for cancer (2). Less attention has been paid to the control of endothelial cell apoptosis during development, although it has been proposed to explain the overall decrease in microvascular density associated with microvascular remodeling.Much of the classical work on microvascular remodeling has been done in the developing retina because of its planar architecture and accessibility, because the expansion and remodeling of this vascular bed is postnatal. A series of elegant studies have clearly established VEGF-A as a driver of vascular expansion in this organ in both development (3, 4) and pathological neovascularization (5-7) as well as a regulator of endothelial cell survival during the remodeling process (8). In the retina, VEGF-A is provided largely by the astrocytes, which enter the retina through the optic disk and spread radially toward the periphery in advance of blood vessel formation (3, 9). The astrocytes are exquisitely sensitive to oxygen levels and strongly induce VEGF-A in hypoxic regions of the retina (10, 11). As the astrocytes and expand radially across the retina, they attract the endothelium to follow and lay a scaffold on which blood vessels form, in part due to the molecular attraction of VEGFR-2 expressed on endothelial cell filopodia with sequestered VEGF on the astrocyte surface (9). Once the new blood vessels bring oxygen to the formerly hypoxic regions of the retina, local VEGF-A levels drop. These events initiate the remodeling process where vessel regression is coordinated with pericyteinduced stabilization (12,13). This coordination of oxygeninduced VEGF-A reduction and pericyte stabilization has led to the hypothesis that the balance in survival sig...

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BackgroundLXRs (Liver X Receptor α and β) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds.ObjectiveBlood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators.MethodsTranscript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRα, LXRβ, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated ex vivo with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623.ResultsA single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRα and LXRβ, and all cell types significantly increased ABCA1 and ABCG1 expression upon ex vivo LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription.ConclusionPeripheral blood cells express LXRα and LXRβ, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anticancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.