Nanoparticulate system for efficient gene transfer into refractory cell targets

A biocompatible, nanoparticulate formulation has been designed to retain, protect, and deliver adenoviral gene constructs over an extended time course. Such devices can be administered locally or systemically with low toxicity. A multipolymeric nanoparticulate system, featuring very high stability in physiologic media, was designed to allow efficient in vitro gene transfer. The efficacy of nanoparticulate delivery is effective in cell systems that are normally refractory to gene transfer, such as pancreatic islets and antigen-presenting cells. The findings suggest a nonspecific uptake system that permits adenoviral particle release within the transfected cells. A comparison with literature data revealed that our system is efficient at much lower levels (at least three orders of magnitude) of infectious viral particles.     

Water-based nanoparticulate polymeric system for protein delivery: permeability control and vaccine application

The idea of using polymeric nanoparticles as drug carriers is receiving an increasing amount of attention both in academia and industry, Nanoparticles have a number of potential applications in protein, drug and vaccine delivery, as well as gene therapy applications. In this article, we focus on this unique drug delivery technology as a method to control the release rate of substances, not only for protein delivery but also for delivering an experimental vaccine immunogen. Nanoparticles were assembled on the basis of ionic interaction between water-soluble polymers so that the resulting particles were stable in physiologic media. Among the typical polymers used to assemble nanoparticles, different polysaccharides, natural amines, and poly-amines were investigated. The entrapped substances tested included a protein and antigens. Polydextran aldehyde was incorporated into the particle core, to enable physiologic cross-linking as a method to control permeability. This resulted in long-term retention of substances that would otherwise rapidly leak out of the nanoparticles. Results of cross-linking experiments clearly demonstrated that the release rate could be substantially reduced, depending on the degree of cross-linking. For vaccine antigen delivery tests, we measured an antibody production after subcutaneous and oral administration. The data indicated that only the cross-linked antigen was immunogenic when the oral route of administration was used. The data presented in this article address primarily the utility of nanoparticulates for oral delivery of vaccine antigen.     

In developing Drosophila neurones the production of gamma-amino butyric acid is tightly regulated downstream of glutamate decarboxylase translation and can be influenced by calcium

The presented work pioneers the embryonic Drosophila CNS for studies of the developmental regulation and function of gamma-amino butyric acid (GABA). We describe for the first time the developmental pattern of GABA in Drosophila and address underlying regulatory mechanisms. Surprisingly, and in contrast to vertebrates, detectable levels of GABA occur late during Drosophila neurogenesis, after essential neuronal proliferation and growth have taken place and synaptogenesis has been initiated. This timeline is almost unchanged when the GABA synthetase glutamate decarboxylase (GAD) is strongly misexpressed throughout the nervous system suggesting a tight post-translational regulation of GABA expression. We confirmed such GABA control mechanisms in an independent model system, i.e. primary Drosophila cell cultures raised in elevated [K+]. The data suggest that, in both systems, GABA suppression occurs via control of GAD activity. Using developing embryos and cell cultures as parallel assay systems for pharmacological and genetic studies we show that the negative regulation of GAD can be overridden by drugs known to elevate intracellular free [Ca2+]. Our results provide the basis for investigations of genetic mechanisms underlying the observed phenomenon, and we discuss the potential implications of this work for Drosophila neurogenesis but also for a general understanding of GAD regulation.     

Requirement of PEN-2 for stabilization of the presenilin N-/C-terminal fragment heterodimer within the gamma-secretase complex

Gamma-secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated beta-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-DeltaC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-DeltaC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the gamma-secretase complex. In addition, expression of PEN-2-DeltaC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Deltaexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for gamma-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the gamma-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for gamma-secretase complex maturation independent of its activity in PS1 endoproteolysis.     

Cigarette smoke-induced accumulation of lung dendritic cells is interleukin-1α-dependent in mice

Background

Evidence suggests that dendritic cells accumulate in the lungs of COPD patients and correlate with disease severity. We investigated the importance of IL-1R1 and its ligands IL-1α and β to dendritic cell accumulation and maturation in response to cigarette smoke exposure.

Methods

Mice were exposed to cigarette smoke using a whole body smoke exposure system. IL-1R1-, TLR4-, and IL-1α-deficient mice, as well as anti-IL-1α and anti-IL-1β blocking antibodies were used to study the importance of IL-1R1 and TLR4 to dendritic cell accumulation and activation.

Results

Acute and chronic cigarette smoke exposure led to increased frequency of lung dendritic cells. Accumulation and activation of dendritic cells was IL-1R1/IL-1α dependent, but TLR4- and IL-1β-independent. Corroborating the cellular data, expression of CCL20, a potent dendritic cells chemoattractant, was IL-1R1/IL-1α-dependent. Studies using IL-1R1 bone marrow-chimeric mice revealed the importance of IL-1R1 signaling on lung structural cells for CCL20 expression. Consistent with the importance of dendritic cells in T cell activation, we observed decreased CD4⁺ and CD8⁺ T cell activation in cigarette smoke-exposed IL-1R1-deficient mice.

Conclusion

Our findings convey the importance of IL-1R1/IL-1α to the recruitment and activation of dendritic cells in response to cigarette smoke exposure.     

Visual versus Automated Evaluation of Chest Computed Tomography for the Presence of Chronic Obstructive Pulmonary Disease

Background

Incidental CT findings may provide an opportunity for early detection of chronic obstructive pulmonary disease (COPD), which may prove important in CT-based lung cancer screening setting. We aimed to determine the diagnostic performance of human observers to visually evaluate COPD presence on CT images, in comparison to automated evaluation using quantitative CT measures.

Methods

This study was approved by the Dutch Ministry of Health and the institutional review board. All participants provided written informed consent. We studied 266 heavy smokers enrolled in a lung cancer screening trial. All subjects underwent volumetric inspiratory and expiratory chest computed tomography (CT). Pulmonary function testing was used as the reference standard for COPD. We evaluated the diagnostic performance of eight observers and one automated model based on quantitative CT measures.

Results

The prevalence of COPD in the study population was 44% (118/266), of whom 62% (73/118) had mild disease. The diagnostic accuracy was 74.1% in the automated evaluation, and ranged between 58.3% and 74.3% for the visual evaluation of CT images. The positive predictive value was 74.3% in the automated evaluation, and ranged between 52.9% and 74.7% for the visual evaluation. Interobserver variation was substantial, even within the subgroup of experienced observers. Agreement within observers yielded kappa values between 0.28 and 0.68, regardless of the level of expertise. The agreement between the observers and the automated CT model showed kappa values of 0.12–0.35.

Conclusions

Visual evaluation of COPD presence on chest CT images provides at best modest accuracy and is associated with substantial interobserver variation. Automated evaluation of COPD subjects using quantitative CT measures appears superior to visual evaluation by human observers.     

Cellular impact and selectivity of half-sandwich organorhodium(III) anticancer complexes and their organoiridium(III) and trichloridorhodium(III) counterparts

Half-sandwich organorhodium(III) complexes and their trichloridorhodium(III) counterparts are potent anticancer agents that enhance the formation of reactive oxygen species and invoke a strong induction of apoptosis in leukemia cells. The antiproliferative activity towards human MCF-7 and HT-29 adenocarcinoma cells of novel nonintercalating complexes containing the 5-substituted phenanthroline ligands 5,6-dimethylphenanthroline, 5-chlorophenanthroline, and 5-nitrophenanthroline (phen*) increases dramatically in the order [(η(5)-C(5)Me(5))IrCl(phen*)](CF(3)SO(3)) < [(η(5)-C(5)Me(5))RhCl(phen*)](CF(3)SO(3)) < mer-[RhCl(3)(DMSO)(phen*)] (DMSO is dimethyl sulfoxide). Improved activity was also achieved by attaching a cell-penetrating peptide to the dipyrido[3,2-a:2',3'-c]phenazine (dppz) ligand of an organorhodium(III) complex. Whereas 5-substitution led to significant improvements in the activity of the organoiridium(III) and trichloridorhodium(III) compounds in comparison with the parent phenanthroline complex, the IC(50) values of their organorhodium(III) counterparts remained effectively invariable. The high activities of the trichloridorhodium(III) complexes (IC(50) = 0.06-0.13 μM) were accompanied by pronounced selectivity towards human cancer cells in comparison with immortalized HEK-293 cells. In contrast, [(η(5)-C(5)Me(5))RhCl(5,6-Me(2)phen)](CF(3)SO(3)) (phen is phenanthroline) was markedly more active towards BJAB lymphoma cells than ex vivo healthy leukocytes and caused an immediate decrease in cellular adhesion possibly associated with interactions with membrane proteins. Its dppz analogue invoked an initial increase in glycolysis to compensate for reduced respiration before inducing a delayed onset of cell death. Strong antimitochondrial activity with respiration impairment and release of cytochrome c was established for both complexes.     

Age,Experience and Sex – Do Female Bulb Mites Prefer Young Mating Partners?

In species where advancing sire age is associated with decreased progeny fitness, female resistance to mating with old partners can be expected to evolve. In polyandrous species, such resistance may be contingent on female mating experience: virgins should be relatively indiscriminate to ensure egg fertility, whereas non‐virgins can be expected to base their re‐mating decisions on the age of their previous versus potential new partners, and ‘trade‐up’ if previously mated with old males. Here, we tested these predictions using a promiscuous and relatively long‐living bulb mite (Rhizoglyphus robini), in which old sire age is associated with decreased fecundity of daughters. In a fully factorial design, we applied two male treatments, young and old, and three female treatments, virgin, previously mated to an old male and previously mated to a young male. Consistent with earlier studies, we observed a reduced mating success of old males. However, we found no support for attributing this result to female discrimination, as female behavior in response to male mounting attempts was not affected by the age of the suitor, or by its interaction with the age of the female’s previous mate. Interestingly, females were passive during 93% of male mounting attempts observed, suggesting that once they are located by a male, they exert little control over copulation. Old males had lower mate‐searching activity and were less efficient in obtaining matings (lower success rate per mounting attempt), suggesting a decreased mate‐securing ability because of aging. Overall, our results suggest that in bulb mites, male ability to secure mates declines with age, whereas they do not support the prediction that females actively discriminate against old partners.     

Leptin and leptin receptor: Analysis of a structure to function relationship in interaction and evolution from humans to fish

Leptin is a circulating protein which regulates dietary intake through binding the leptin receptor. Numerous labs have used known structures and mutagenesis to study this binding process in common animal models (human, mouse and rat). Understanding this binding process in other vertebrate species will allow for a better understanding of leptin and leptin receptor function. The binding site between leptin and leptin receptor is highly conserved in mammals as confirmed through sequence alignments mapped onto structures of both leptin and leptin receptor. More variation in this interaction is found in lizard and frog sequences. Using our models, we show that the avian leptin sequences have far less variation in the binding site than does the leptin receptor. This analysis further suggests that avian leptins are artifactual. In fish, gene duplication events have led to the expression of multiple leptin proteins. These multiple leptin proteins have variation in the regions interacting with leptin receptor. In zebrafish and the Japanese rice fish, we propose that leptin A has a higher binding energy than does B. Differing binding energies are evidence of either divergent functions, different binding confirmations, or other protein partners of leptin B.