Rescue of the mouse DDK syndrome by parent-of-origin-dependent modifiers

When females of the DDK inbred mouse strain are mated to males of other strains, 90-100% of the resulting embryos die during early embryonic development. This DDK syndrome lethality results from incompatibility between an ooplasmic DDK factor and a non-DDK paternal gene, which map to closely linked loci on chromosome 11. It has been proposed that the expression of the gene that encodes the ooplasmic factor is subject to allelic exclusion in oocytes. Previous studies have demonstrated the existence of recessive modifiers that increase lethality in the C57BL/6 and BALB/c strains. These modifiers are thought to skew the choice of allele undergoing allelic exclusion in the oocytes of heterozygous females. In the present study, we demonstrate the presence of modifiers in three Mus musculus domesticus wild-derived strains, PERA, PERC, and RBA. These modifiers completely rescued DDK syndrome lethality. We mapped the major locus that is responsible for rescue in PERA and PERC crosses to proximal chromosome 13 and named this locus Rmod1 (Rescue Modifier of the DDK Syndrome 1). Our experiments demonstrate that PERA or PERC alleles at Rmod1 rescue lethality independently of allelic exclusion. In addition, rescue of the lethal phenotype depends on the parental origin of the Rmod1 alleles; transmission through the dam leads to rescue, while transmission through the sire has no effect.     

Triggering of dendritic cell responses after exposure to activated, but not resting, apoptotic PBMCs

Dendritic cells (DCs) can be activated by signaling via pathogen receptors, by interaction with activated T cells or by exposure to inflammatory mediators. Clearance of apoptotic cells by DCs is generally considered a silent event that is not associated with an inflammatory response. Necrotic cell death, in contrast, leads to induction of inflammation. However, emerging data challenge the view of apoptotic cells as inherently nonimmunogenic. In this study, we report that the activation state of the apoptotic cell may determine whether the exposed DC becomes activated and rendered proficient in Ag presentation. We show that coculture with activated, but not resting, apoptotic PBMCs leads to up-regulation of surface expression of the costimulatory molecules CD80, CD83, and CD86 in human DCs as well as release of proinflammatory cytokines. Furthermore, we show that DCs exposed to allogeneic, activated apoptotic PBMCs induce proliferation and IFN-gamma production in autologous T cells. Together, these findings show that activated apoptotic PBMCs per se provide an activation/maturation signal to DCs, suggesting that activated apoptotic PBMCs possess endogenous adjuvant properties.     

Fibrin gel-immobilized VEGF and bFGF efficiently stimulate angiogenesis in the AV loop model

The modulation of angiogenic processes in matrices is of great interest in tissue engineering. We assessed the angiogenic effects of fibrin-immobilized VEGF and bFGF in an arteriovenous loop (AVL) model in 22 AVLs created between the femoral artery and vein in rats. The loops were placed in isolation chambers and were embedded in 500 microL fibrin gel (FG) (group A) or in 500 microL FG loaded with 0.1 ng/microL VEGF and 0.1 ng/microL bFGF (group B). After two and four weeks specimens were explanted and investigated using histological, morphometrical, and ultramorphological [scanning electron microscope (SEM) of vascular corrosion replicas] techniques. In both groups, the AVL induced formation of densely vascularized connective tissue with differentiated and functional vessels inside the fibrin matrix. VEGF and bFGF induced significantly higher absolute and relative vascular density and a faster resorption of the fibrin matrix. SEM analysis in both groups revealed characteristics of an immature vascular bed, with a higher vascular density in group B. VEGF and bFGF efficiently stimulated sprouting of blood vessels in the AVL model. The implantation of vascular carriers into given growth factor-loaded matrix volumes may eventually allow efficient generation of axially vascularized, tissue-engineered composites.     

Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH-dependent stability (3 and 4), potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver (t(1/2) = 3.7 min), intestinal (t(1/2) = 12.5 min), and Caco-2 cell homogenates (t(1/2) = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC50 value, 0.68 microM vs 3.0 microM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.     

Endothelin-1 and pancreatic islet vasculature: studies in vivo and on isolated, vascularly perfused pancreatic islets

Endothelin-1 (ET-1) is a potent endothelium-derived vasoconstrictor, which also stimulates insulin release. The aim of the present study was to evaluate whether exogenously administered ET-1 affected pancreatic islet blood flow in vivo in rats and the islet arteriolar reactivity in vitro in mice. Furthermore, we aimed to determine the ET-receptor subtype that was involved in such responses. When applying a microsphere technique for measurements of islet blood perfusion in vivo, we found that ET-1 (5 nmol/kg) consistently and markedly decreased total pancreatic and especially islet blood flow, despite having only minor effects on blood pressure. Neither endothelin A (ET(A)) receptor (BQ-123) nor endothelin-B (ET(B)) receptor (BQ-788) antagonists, alone or in combination, could prevent this reduction in blood flow. To avoid confounding interactions in vivo, we also examined the arteriolar vascular reactivity in isolated, perfused mouse islets. In the latter preparation, we demonstrated a dose-dependent constriction in response to ET-1. Administration of BQ-123 prevented this, whereas BQ-788 induced a right shift in the response. In conclusion, the pancreatic islet vasculature is highly sensitive to exogenous ET-1, which mediates its effect mainly through ET(A) receptors.     

Epidermal growth factor- and stress-induced loss of gap junctional communication is mediated by ERK-1/ERK-2 but not ERK-5 in rat liver epithelial cells

Extracellular signal-regulated kinases (ERK) 1 and 2 as well as ERK-5 were previously suggested to phosphorylate connexin-43 and to contribute to the modulation of gap junctional intercellular communication (GJC). Exposure of rat liver epithelial cells to epidermal growth factor (EGF) or the redox cycling and alkylating agent menadione resulted in phosphorylation of connexin-43 and loss in GJC, both of which were abrogated by pharmacological inhibitors of ERK-1/2 activation, if used in concentrations that selectively abrogate phosphorylation of ERK-1/2 but not of ERK-5. Thus, EGF- or menadione-induced loss of GJC is mediated by ERK-1/2 but not ERK-5 in rat liver epithelial cells.