Drug Delivery of Oligonucleotides at the Blood-Brain Barrier: a Therapeutic Strategy for Inflammatory Diseases of the Central Nervous System

In this project a vector for drug delivery into brain endothelial cells, consisting of an anti-transferrin receptor antibody and a complex of polyethylenimine (PEI) and an oligodeoxynucleotide (ODN) drug was evaluated. NF-kB decoys are short double-stranded ODNs, which contain a recognition sequence for binding to the transcription factor. By binding to the transcription factor NF-kB they prevent the transcription of several genes involved in inflammatory processes, such as adhesion molecules and pro-inflammatory enzymes. Adhesion molecules promote the transmigration of activated T lymphocytes across the blood-brain barrier (BBB), leading to demyelination of nerve fibers within the brain, as seen in inflammatory diseases like Multiple Sclerosis (MS). Inhibition of lymphocyte transmigration by downregulation of the expression of these genes may be therapeutically beneficial. The rat anti mouse anti-transferrin receptor antibody 8D3 was purified from hybridoma supernatant and was conjugated to recombinant streptavidin (SA) in order to bind to biotinylated ligands. In vivo pharmacokinetics and in vitro binding studies were performed to evaluate the 8D3-SA vector. Pharmacokinetic studies showed a comparable half-life and area under the curve (AUC) of antibody conjugate and free 8D3 antibody in plasma. Immunohistochemistry with a brain endothelioma cell line (bEnd5) revealed a similar binding behavior for both, antibody and its streptavidin conjugate. The specificity of uptake of 3H-biotin-8D3SA by bEnd5 cells could be observed in experiments carried out either with an acid wash step to remove all surface bound antibody, or by using the mouse anti rat transferrin receptor antibody OX26. The biotinylated drug applied in this project was a polymer – polyethylenimine (PEI), which is able to complex a double-stranded 20bp ODN sequence (NF-kB decoy). The low molecular weight PEI (LMW PEI) of 2,700 Da was chosen due to such favorable properties as low cytotoxicity, high transfection efficiency, and narrow size distribution. A co-polymer of PEI and biotinylated PEG was synthesized to allow coupling to the vector. Stability studies were carried out to examine the characteristics of bioPEGPEI/ODN complexes in salt solution, under addition of 10% or 20% plasma, or by incubation of complex in medium over a long time frame. Particle size measurements in PBS pH 7.4 revealed an optimal size distribution of bioPEGPEI/NF-kB complex at a ratio PEI-amine/ODN-phosphate of 6:1 with an average particle size of 120nm, which was stable for at least 1 week. Gel retardation assays also showed a complete complexation of bioPEGPEI and ODN at this ratio. The addition of 8D3-SA increased the complex size by 40nm. In vitro pharmacological effects of bioPEGPEI/NF-kB could be observed after treatment of bEnd5 cells with different concentrations of NF-kB decoy over a time frame of 48 hours. An inhibition of VCAM-1 (isoform 1 and 2) expression was already visible after 12 hours with a steady increase of this inhibitory effect to 48 hours. ODN-drug concentrations of 5uM in complex with bioPEGPEI showed a nearly complete suppression of VCAM-1 expression. Coupling of bioPEGPEI/NF-kB decoy to the vector decreased the concentration required for a reduction of VCAM-1 mRNA to 0.5uM - 1uM, suggesting an increase in potency by targeting to the transferring receptor. Pharmacokinetic studies in mice showed the behavior of bioPEGPEI/NF-kB complexes with different N/P ratios. At a N/P ratio of 6:1 the AUC (area under the plasma curve) could be increased 2-fold compared to free NF-kB. A coupling to the targeting vector did not further change the pharmacokinetic characteristics. The drug targeting approach investigated in this project promises to be an excellent model for in vitro applications. The in vivo administration must be further evaluated with the goal to improve the pharmacokinetic behavior (enhance AUC). This may require additional modifications of the bioPEGPEI copolymer

Modellierung der Instabilität von Strömungen über das Drehmomentengleichgewicht in der Wirbelhülle

Turbulenz in Strömungen entsteht aus Wirbeln. Aus geometrischen Parametern der Wirbelhülle lassen sich die kritischen Reynoldszahlen von Plattenströmung und Rohrströmung ermitteln. Die Hysterese der Instabilität wird begründet

Antimicrobial Peptides and Skin: A Paradigm of Translational Medicine

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic peptides with broad-spectrum microbicidal activity against both bacteria and fungi. In mammals, AMPs form the first line of host defense against infections and generally play an important role as effector agents of the innate immune system. The AMP era was born more than 6 decades ago when the first cationic cyclic peptide antibiotics, namely polymyxins and tyrothricin, found their way into clinical use. Due to the good clinical experience in the treatment of, for example, infections of mucus membranes as well as the subsequent understanding of mode of action, AMPs are now considered for treatment of inflammatory skin diseases and for improving healing of infected wounds. Based on the preclinical findings, including pathobiochemistry and molecular medicine, targeted therapy strategies are developed and first results indicate that AMPs influence processes of diseased skin. Importantly, in contrast to other antibiotics, AMPs do not seem to propagate the development of antibiotic-resistant micro-organisms. Therefore, AMPs should be tested in clinical trials for their efficacy and tolerability in inflammatory skin diseases and chronic wounds. Apart from possible fields of application, these peptides appear suited as an example of the paradigm of translational medicine for skin diseases which is today seen as a `two-way road' - from bench to bedside and backwards from bedside to bench. Copyright (c) 2012 S. Karger AG, Base

Observational constraints on atmospheric and oceanic cross-equatorial heat transports: revisiting the precipitation asymmetry problem in climate models

Satellite based top-of-atmosphere (TOA) and surface radiation budget observations are combined with mass corrected vertically integrated atmospheric energy divergence and tendency from reanalysis to infer the regional distribution of the TOA, atmospheric and surface energy budget terms over the globe. Hemispheric contrasts in the energy budget terms are used to determine the radiative and combined sensible and latent heat contributions to the cross-equatorial heat transports in the atmosphere (AHT_EQ) and ocean (OHT_EQ). The contrast in net atmospheric radiation implies an AHT_EQ from the northern hemisphere (NH) to the southern hemisphere (SH) (0.75 PW), while the hemispheric difference in sensible and latent heat implies an AHT_EQ in the opposite direction (0.51 PW), resulting in a net NH to SH AHT_EQ (0.24 PW). At the surface, the hemispheric contrast in the radiative component (0.95 PW) dominates, implying a 0.44 PW SH to NH OHT_EQ. Coupled model intercomparison project phase 5 (CMIP5) models with excessive net downward surface radiation and surface-to-atmosphere sensible and latent heat transport in the SH relative to the NH exhibit anomalous northward AHT_EQ and overestimate SH tropical precipitation. The hemispheric bias in net surface radiative flux is due to too much longwave surface radiative cooling in the NH tropics in both clear and all-sky conditions and excessive shortwave surface radiation in the SH subtropics and extratropics due to an underestimation in reflection by clouds

Organizing Effects of Sex Steroids on Brain Aromatase Activity in Quail

Preoptic/hypothalamic aromatase activity (AA) is sexually differentiated in birds and mammals but the mechanisms controlling this sex difference remain unclear. We determined here (1) brain sites where AA is sexually differentiated and (2) whether this sex difference results from organizing effects of estrogens during ontogeny or activating effects of testosterone in adulthood. In the first experiment we measured AA in brain regions micropunched in adult male and female Japanese quail utilizing the novel strategy of basing the microdissections on the distribution of aromatase-immunoreactive cells. The largest sex difference was found in the medial bed nucleus of the stria terminalis (mBST) followed by the medial preoptic nucleus (POM) and the tuberal hypothalamic region. A second experiment tested the effect of embryonic treatments known to sex-reverse male copulatory behavior (i.e., estradiol benzoate [EB] or the aromatase inhibitor, Vorozole) on brain AA in gonadectomized adult males and females chronically treated as adults with testosterone. Embryonic EB demasculinized male copulatory behavior, while vorozole blocked demasculinization of behavior in females as previously demonstrated in birds. Interestingly, these treatments did not affect a measure of appetitive sexual behavior. In parallel, embryonic vorozole increased, while EB decreased AA in pooled POM and mBST, but the same effect was observed in both sexes. Together, these data indicate that the early action of estrogens demasculinizes AA. However, this organizational action of estrogens on AA does not explain the behavioral sex difference in copulatory behavior since AA is similar in testosterone-treated males and females that were or were not exposed to embryonic treatments with estrogens

Effects of episodic future thinking and self-projection on children’s prospective memory performance

The present study is the first to investigate the benefits of episodic future thinking (EFT) at encoding on prospective memory (PM) in preschool (age: M = 66.34 months, SD = 3.28)and primary school children (age: M = 88.36 months, SD = 3.12). A second aim was to examine if self-projection influences the possible effects of EFT instructions. PM was assessed using a standard PM paradigm in children with a picture-naming task as the ongoing activity in which the PM task was embedded. Further, two first- and two second-order ToM tasks were administered as indicator of children’s self-projection abilities. Forty-one preschoolers and 39 school-aged children were recruited. Half of the participants in each age group were instructed to use EFT as a strategy to encode the PM task, while the others received standard PM instructions. Results revealed a significant age effect, with school-aged children significantly outperforming preschoolers and a significant effect of encoding condition with overall better performance when receiving EFT instructions compared to the standard encoding condition. Even though the interaction between age group and encoding condition was not significant, planned comparisons revealed first evidence that compared to the younger age group, older children’s PM benefited more from EFT instructions during intention encoding. Moreover, results showed that although self-projection had a significant impact on PM performance, it did not influence the effects of EFT instructions. Overall, results indicate that children can use EFT encoding strategies to improve their PM performance once EFT abilities are sufficiently developed. Further, they provide first evidence that in addition to executive functions, which have already been shown to influence the development of PM across childhood, self-projection seems to be another key mechanism underlying this development

Geodesy and metrology with a transportable optical clock

partially_open24openGrotti, Jacopo; Koller, Silvio; Vogt, Stefan; Häfner, Sebastian; Sterr, Uwe; Lisdat, Christian; Denker, Heiner; Voigt, Christian; Timmen, Ludger; Rolland, Antoine; Baynes, Fred N.; Margolis, Helen S.; Zampaolo, Michel; Thoumany, Pierre; Pizzocaro, Marco; Rauf, Benjamin; Bregolin, Filippo; Tampellini, Anna; Barbieri, Piero; Zucco, Massimo; Costanzo, Giovanni A.; Clivati, Cecilia; Levi, Filippo; Calonico, DavideGrotti, Jacopo; Koller, Silvio; Vogt, Stefan; Häfner, Sebastian; Sterr, Uwe; Lisdat, Christian; Denker, Heiner; Voigt, Christian; Timmen, Ludger; Rolland, Antoine; Baynes, Fred N.; Margolis, Helen S.; Zampaolo, Michel; Thoumany, Pierre; Pizzocaro, Marco; Rauf, Benjamin; Bregolin, Filippo; Tampellini, Anna; Barbieri, Piero; Zucco, Massimo; Costanzo, Giovanni A.; Clivati, Cecilia; Levi, Filippo; Calonico, David

Synergistic ecoclimate teleconnections from forest loss in different regions structure global ecological responses

ABSTRACT: Forest loss in hotspots around the world impacts not only local climate where loss occurs, but also influences climate and vegetation in remote parts of the globe through ecoclimate teleconnections. The magnitude and mechanism of remote impacts likely depends on the location and distribution of forest loss hotspots, but the nature of these dependencies has not been investigated. We use global climate model simulations to estimate the distribution of ecologically-relevant climate changes resulting from forest loss in two hotspot regions: western North America (wNA), which is experiencing accelerated dieoff, and the Amazon basin, which is subject to high rates of deforestation. The remote climatic and ecological net effects of simultaneous forest loss in both regions differed from the combined effects of loss from the two regions simulated separately, as evident in three impacted areas. Eastern South American Gross Primary Productivity (GPP) increased due to changes in seasonal rainfall associated with Amazon forest loss and changes in temperature related to wNA forest loss. Eurasia’s GPP declined with wNA forest loss due to cooling temperatures increasing soil ice volume. Southeastern North American productivity increased with simultaneous forest loss, but declined with only wNA forest loss due to changes in VPD. Our results illustrate the need for a new generation of local-to-global scale analyses to identify potential ecoclimate teleconnections, their underlying mechanisms, and most importantly, their synergistic interactions, to predict the responses to increasing forest loss under future land use change and climate change

Eliminating Rabies in Estonia

The compulsory vaccination of pets, the recommended vaccination of farm animals in grazing areas and the extermination of stray animals did not succeed in eliminating rabies in Estonia because the virus was maintained in two main wildlife reservoirs, foxes and raccoon dogs. These two species became a priority target therefore in order to control rabies. Supported by the European Community, successive oral vaccination (OV) campaigns were conducted twice a year using Rabigen® SAG2 baits, beginning in autumn 2005 in North Estonia. They were then extended to the whole territory from spring 2006. Following the vaccination campaigns, the incidence of rabies cases dramatically decreased, with 266 cases in 2005, 114 in 2006, four in 2007 and three in 2008. Since March 2008, no rabies cases have been detected in Estonia other than three cases reported in summer 2009 and one case in January 2011, all in areas close to the South-Eastern border with Russia. The bait uptake was satisfactory, with tetracycline positivity rates ranging from 85% to 93% in foxes and from 82% to 88% in raccoon dogs. Immunisation rates evaluated by ELISA ranged from 34% to 55% in foxes and from 38% to 55% in raccoon dogs. The rabies situation in Estonia was compared to that of the other two Baltic States, Latvia and Lithuania. Despite regular OV campaigns conducted throughout their territory since 2006, and an improvement in the epidemiological situation, rabies has still not been eradicated in these countries. An analysis of the number of baits distributed and the funding allocated by the European Commission showed that the strategy for rabies control is more cost-effective in Estonia than in Latvia and Lithuania