Assessing cytotoxicity of (iron oxide‐based) nanoparticles: an overview of different methods exemplified with cationic magnetoliposomes

Iron oxide nanoparticles are the most widely used T₂/T₂* contrast agents and for biomedical research purposes, one of the main applications is the in vitro labeling of stem or therapeutic cells, allowing them to be subsequently tracked in vivo upon transplantation. To allow this, the nanoparticles used should not show any sign of cytotoxicity and not affect cellular physiology as this could impede normal cell functionality in vivo or lead to undesired side‐effects. Assessing the biocompatibility of the nanoparticles has proven to be quite a difficult task. In the present work, a small overview of commonly used assays is presented in order to assess several aspects, such as cell viability, induction of reactive oxygen species, nanoparticle uptake, cellular morphology, cellular proliferation, actin cytoskeleton architecture and differentiation of stem cells. The main focus is on comparing the advantages and disadvantages of the different assays, highlighting several common problems and presenting possible solutions to these problems as well as pointing out the high importance of the relationship between intracellular nanoparticle concentration and cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Measuring attentional bias to threat in children and adolescents: A matter of speed?

Attentional bias to threat has proven a robust phenomenon in adults but has shown to be less stable in children. This study investigated whether a generally slow response speed may potentially obscure the effects of threat upon attention in children. Three non-clinical samples of different age (children, adolescents, young adults) performed an emotional modification of the spatial cueing task. Attentional bias was examined by comparing cueing effects by fear-conditioned cues (CS+) and neutral cues (CS−). Overall, analyses showed no biased attention to threat, and no differences between age groups could be detected. Further analyses indicated that the inability to demonstrate attentional bias to threat in the present study was due to the large response speed variability in our sample. Quintile analyses of the reaction times revealed that an overall attentional bias to threat across all groups was present when only the fast reaction times were taken into account. Methodological implications for measuring attentional bias in young age groups are discussed.     

Mandibuläre Hypoplasie

In order to restore function and ¶esthetics in mandibular hypoplasia, transplantation of osteochondral grafts and the distraction osteogenesis technique are used alternatively. In a retrospective study the indication, growth and complication rate ¶of osteochondral graft and distraction osteogenesis cases in mandibular hypoplasia were compared. In total, 12 patients (4–14 years old; 7 female, 5 male) with different grades of mandibular hypoplasia received osteochondral grafts (7 costochondral, 5 iliac crest; observation period 4–9 years). In 5 patients (10–12 years old; 1 female, 4 male), distraction osteogenesis was performed (observation period 10 months to 3 1/2 years). In the results, according to the literature, the range of growth after osteochondral grafting varied considerably, approximate normal growth was noticed in only two cases. On the other hand, all patients treated by distraction osteogenesis showed a stable increase of bone length as expected. After osteochondral grafts, adequate growth is to be expected in the minority of cases and it does not seem predictable in individual cases. Therefore, the indication for osteochondral grafts should be restricted to severe hypoplasia, where the local amount of bone does not allow the use of a distraction device, and to cases where support of the mandibula at the skull base is necessary. In all other cases of mandibular hypoplasia in children, distraction osteogenesis should be discussed as the primary option, even if in the long term a second surgical procedure should be necesssary.     

A re-evaluation of k0 and related nuclear data for the 555.8 keV gamma-line emitted by the Rh-Rh mother–daughter pair for use in NAA

A re-evaluation is made of the k₀-factor and related nuclear data for the 555.8 keV gamma-ray of the ^104mRh-¹⁰⁴Rh mother–daughter pair that are important in neutron activation analysis (NAA). This study considers that the relevant level is also fed by the 4.34 min ^104mRh mother (with an absolute gamma-ray emission probability γ₂=0.13%) and not only, as assumed in former work, by the 42.3 s ¹⁰⁴Rh daughter isotope (with γ₃=2.0%). In view of this, generalised equations were developed for both the experimental determination and the analytical use of the k₀-factor and of the associated parameters k₀(m)/k₀(g), Q₀(m) and Q₀(g) [(m):^104mRh; (g): ¹⁰⁴Rh], requiring the introduction of the γ₂ and γ₃ data and also of the ^104mRh→¹⁰⁴Rh fractional decay factor F₂(=0.9987). The experimental determinations were based on irradiations performed in the BR1 reactor in Mol and the WWR-M reactor in Budapest. Furthermore, considering the special formation of the 555.8 keV gamma-ray, the procedure for true-coincidence correction was revised as well. All this led to the compilation and recommendation of a new set of ‘k₀-NAA’ data.     

Vaccination of turkeys against Chlamydophila psittaci through optimised DNA formulation and administration

We have demonstrated that vaccination of turkeys with an unformulated DNA vaccine induces significant protection against Chlamydophila (Cp.) psittaci infections. Nevertheless, the immunogenicity of the DNA vaccine can still be improved by increasing translation and transfection efficiency. Therefore, the ompA codon was adapted to the codon usage in birds, resulting in pcDNA1/MOMP_opt. To increase gene transfer, polyplexes of pcDNA1/MOMP_opt–EGFP with different cationic polymers, such as linear and branched polyethyleneimine (lPEI and brPEI) and starburst PAMAM dendrimers, and lipoplexes with cationic DOTAP/DOPE liposomes were created. Transfection of lPEI and brPEI polyplexes with an N/P ratio of 8 resulted in the highest transfection efficiencies, but lPEI polyplexes were completely destroyed following nebulisation. Secondly, we examined the capacity of nebulised or intramuscularly (IM) administered brPEI-pcDNA1/MOMP_opt to induce a significant protective immune response in SPF turkeys experimentally infected with 10⁸ TCID₅₀ of a virulent Cp. psittaci strain. Results were compared to IM administration of naked plasmid DNA and to results of non-vaccinated animals. Intramuscular administration of brPEI-pcDNA1/MOMP_opt increased the immunogenicity of the Cp. psittaci DNA vaccine as compared to IM administration of pcDNA1/MOMP_opt or aerosol delivery of brPEI-pcDNA1/MOMP_opt. Improved immunogenicity was correlated with increased protection. Vaccinated groups were significantly protected against Cp. psittaci challenge.     

A pyogenic, ruptured Baker’s cyst induced by arthroscopic pressure pump irrigation

Post-steroid septic arthritis can be treated with irrigation pump assisted arthroscopic synovectomy. The high-intra-articular fluid pressures can force the pyogenic fluid into a pre-existing Baker’s cyst. The cyst can rupture and with the pre-existing steroid induced immune-suppression, the calf abscess will be hard to control. Therefore, thorough investigation with an ultrasound-guided aspiration followed by an early drainage of the collection is warranted and mandatory. Close monitoring for the development of a deep thrombosis of the popliteal vein is required.     

Experimental performance assessment of SPM for SPECT neuroactivation studies using a subresolution sandwich phantom design

The validity domain of voxel-based statistical analysis of SPECT neuroactivation studies with statistical parametric mapping (SPM) has been investigated by a limited number of theoretical and simulation studies. In this work, an experimental setup is described with an assessment of its activation detection performance together with the influence of acquisition and processing parameters. A subresolution sandwich phantom was constructed using a printed high-resolution digital Hoffman phantom with a (99m)TcO(4)-ink mixture. Activations of 8, 16, and 24 mm diameter, with an intensity ranging from 5 to 60%, were constructed in the right frontal cortex, anterior and posterior cingulate, and left striatum. Triple-headed SPECT acquisitions were carried out using both fan-beam and parallel beam geometry. The impact of activation characteristics (size, intensity and location), study size, physical degradation factors, and reconstruction technique were studied using SPM99 in a group comparison design with correction for multiple comparisons. For a 15 x 15 design, all 24-mm activations of 5% intensity were detected for the fan-beam data. Decreased focus or study size, lower SPECT resolution, absence of scatter, and attenuation correction resulted in an increase in minimally detectable activation. For a single study referred to 15 control studies, only 24-mm activation foci with a minimal intensity of 10% were detected in the optimal configuration. This approach allows experimental parameter optimization of SPM-based group or single-subject SPECT activation studies compared to normal data, as used in clinical applications. In principle, these findings can be extended to SPECT receptor studies or PET data.     

The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type.

Understanding cellular uptake and intracellular processing of nonviral gene delivery systems is a key aspect in developing more efficient vectors. In this study, the impact of clathrin- and caveolae/lipid-raft-dependent endocytosis on cell entry and overall transfection efficiency of polyethylenimine (PEI) polyplexes was evaluated. Most remarkably, the internalization pathway mediating successful transfection depended on both cell type and polyplex type applied. Colocalization studies with transferrin and cholera toxin B revealed that at least two specific endocytosis pathways--the clathrin-dependent and the lipid-raft-dependent--mediated cellular uptake of PEI polyplexes. With the help of specific uptake inhibitors (chlorpromazine and filipin III), cell-line-dependent variations regarding the route of successful transfection were observed (HUH-7, COS-7, HeLa). In COS-7 cells, the clathrin-dependent pathway was the main contributor to the transfection process. In HUH-7 cells, gene transfer by linear PEI polyplexes succeeded mainly via the clathrin-dependent route, whereas transfection by branched PEI polyplexes was mediated by both pathways. In HeLa cells, both pathways were able to mediate successful gene delivery. However, the lipid-raft-dependent pathway was more relevant. The study also revealed that the concentration window between specific inhibitory function and nonspecific toxicity of the uptake inhibitors was very narrow.