Sphingosine kinase (SphK) is overexpressed by a variety of cancers, and its phosphorylation of sphingosine results in accumulation of sphingosine-1-phosphate (S1P) and activation of anti-apoptotic signal transduction. Existing data indicate a role for S1P in viral pathogenesis, but roles for SphK and S1P in virus-associated cancer progression have not been defined. Rare pathologic variants of diffuse large B-cell lymphoma arise preferentially in the setting of HIV infection, including primary effusion lymphoma (PEL), a highly mortal tumor etiologically linked to the Kaposi’s sarcoma-associated herpesvirus (KSHV). We have found that ABC294640, a novel clinical-grade small molecule selectively targeting SphK (SphK2 >> SphK1), induces dose-dependent caspase cleavage and apoptosis for KSHV+ patient-derived PEL cells, in part though inhibition of constitutive signal transduction associated with PEL cell proliferation and survival. These results were validated with induction of PEL cell apoptosis using SphK2-specific siRNA, as well as confirmation of drug-induced SphK inhibition in PEL cells with dose-dependent accumulation of pro-apoptotic ceramides and reduction of intracellular S1P. Furthermore, we demonstrate that systemic administration of ABC294640 induces tumor regression in an established human PEL xenograft model. Complimentary ex vivo analyses revealed suppression of signal transduction and increased KSHV lytic gene expression within drug-treated tumors, with the latter validated in vitro through demonstration of dose-dependent viral lytic gene expression within PEL cells exposed to ABC294640. Collectively, these results implicate interrelated mechanisms and SphK2 inhibition in the induction of PEL cell death by ABC294640 and rationalize evaluation of ABC294640 in clinical trials for the treatment of KSHV-associated lymphoma.

There is a controversy about the biocompatibility of silicon nitride ceramics contained in the literature, which appears to be related to a factor of the individual chemical composition of different qualities of silicon nitride ceramics and of the different surface properties. This study attempts to investigate the cytotoxicity of different qualities of industrial silicon nitride ceramics applying an L929-cell culture model in a direct contact assay combined with a cell viability assessment. Five different qualities of industrial standard silicon nitride ceramics were chosen for in vitro testing. The chemical composition was determined by EDS analysis. Different biomedically approved aluminium oxide qualities, a titanium alloy, glass and polyvinylchloride (PVC) served as control materials. L929 mice fibroblasts were incubated directly on the materials for 24 h, stained with bisbenzimide and propidium iodine for double fluorochromasia viability testing, and evaluated by inversion-fluorescence microscopy to control cell morphology, viability and cell counts compared to empty well values. Scanning electron microscopy was applied to additionally investigate cell morphology. There was no observation of cytotoxic effects on the silicon nitride ceramic samples; moreover cell morphology remained the same as on aluminium oxide and titanium. Viability testing revealed the presence of avital cells exclusively on PVC, which served as a negative control. Cell counts on all polished surfaces showed significantly higher numbers, whereas some rough surface samples showed significantly lower numbers. We conclude that silicon nitride ceramics show no cytotoxic effects and should be considered for biomedical application owing to its favourable physiochemical properties, especially its superior resistance to mechanical stress, which would be useful for compression loaded conditions. Polished surfaces would appear to promote advanced biocompatibility.

We have utilized protective oligonucleotides to modify DNA fragments with osmium tetroxide complexes without compromising their ability to hybridize with immobilized thiol-linked probe-SAMs on gold electrodes. Due to reversible voltammetric signals of Os(VI/IV), this method allowed sensitive electrochemical hybridization detection of short (25 bases) and long (120 bases) thymine-containing DNA targets. The detection limit was 3.2 nM for the long target. We found an optimum 40 degrees C hybridization temperature for the short target. No interference by noncomplementary DNA was observed. At least 10 repetitive hybridization experiments at the same probe-SAM were possible with thermal denaturation in between. Such use of protective strands could be useful also for other types of DNA recognition and even for other DNA-modifying agents. Moreover, it is possible to produce electrochemically active oligonucleotides (targets and reporter probes) in ones own laboratory in a simple way.

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This paper reports about the influence of temperature, hybridization time and convection upon the detection of osmium tetroxide bipyridine-labeled target oligonucleotides at rotating gold disk (RDE) and heated low temperature co-fired ceramics (LTCC) gold disk electrodes. We used mixed self-assembled monolayers of hexathiol-linked probe oligonucleotides and mercaptohexanol on the gold surface of the electrodes for the hybridization detection of the labeled targets by means of square-wave voltammetry. Due to protective strands, the osmium tetroxide-modified target strands were still able to hybridize with the immobilized probe strands. The hybridization of such osmium tetroxide bipyridine-modified target strands with thiol-linked probe strands immobilized on gold yielded large reversible square-wave-voltammetric signals. Rotation speed and, hence, mass transport due to convection has only marginal effects. On the other hand, temperature affects greatly the hybridization step as indicated by both heated LTCC electrode in cold and RDE in warm hybridization solution. Calculated detection limits of 3.6 and 3.1 nM targets at the RDE and the LTCC electrode, respectively, have been almost the same at both types of electrodes. Applying an appropriate temperature during hybridization is more important than mechanically enhanced mass transport.

Within the context of novel stent designs we developed a dual drug-eluting stent (DDES) with an abluminally focussed release of the potent anti-proliferative drug sirolimus and a luminally focussed release of atorvastatin with stabilizing effect on atherosclerotic deposits and stimulating impact on endothelial function, both from biodegradable poly(L-lactide)-based stent coatings. With this concept we aim at simultaneous inhibition of in-stent restenosis as a result of disproportionally increased smooth muscle cell proliferation and migration as well as thrombosis due to failed or incomplete endothelialisation. The especially adapted spray-coating processes allowed the formation of smooth form-fit polymer coatings at the abluminal and luminal side with 70% respectively 90% of the drug/polymer solution being deposited at the intended stent surface. The impacts of tempering, sterilization, and layer composition on drug release are thoroughly discussed making use of a semi-empirical model. While tempering at 80 °C seems to be necessary for the achievement of adequate and sustained drug release, the coating sequence for DDES should be rather abluminal-luminal than luminal-abluminal, as reduction of the amount of sirolimus eluted luminally could then potentially minimize the provocation of endothelial dysfunction. In vitro proliferation and viability assays with smooth muscle and endothelial cells underline the high potential of the developed DDES.

Drug-coated balloons are medical devices designed to locally deliver drug to diseased segments of the vessel wall. For these dosage forms, drug transfer to the vessel wall needs to be examined in detail, since drug released into the blood is cleared from the site. In order to examine drug transfer, a new in vitro setup was developed combining the estimation of drug loss during advancement to the site of application in a model coronary artery pathway with a hydrogel compartment representing, as a very simplified model, the vessel wall. The transfer of fluorescent model substances as well as the drug paclitaxel from coated balloons to the simulated vessel wall was evaluated using this method. The model was suitable to quantify the fractions transferred to the hydrogel and also to qualitatively assess distribution patterns in the hydrogel film. In the case of fluorescein sodium, rhodamin b and paclitaxel, vast amounts of the coated substance were lost during the simulated passage and only very small fractions of about 1% of the total load were transferred to the gel. This must be attributed to good water solubility of the fluorescent substances and the mechanical instability of the paclitaxel coating. Transfer of the hydrophobic model substance triamterene was however nearly unaffected by the preliminary tracking procedure with transferred fractions ranging from 8% to 14%. Analysis of model substance distribution yielded inhomogeneous distributions indicating that the coating was not evenly distributed on the balloon surface and that a great fraction of the coating liquid did not penetrate the folds of the balloon. This finding is contradictory to the generally accepted assumption of a drug depot inside the folds and emphasizes the necessity to thoroughly characterize in vitro performance of drug-coated balloons to support the very promising clinical data.