Omnisphero: a high-content image analysis (HCA) approach for phenotypic developmental neurotoxicity (DNT) screenings of organoid neurosphere cultures in vitro.

Current developmental neurotoxicity (DNT) testing in animals faces major limitations, such as high cost and time demands as well as uncertainties in their methodology, evaluation and regulation. Therefore, the use of human-based 3D in vitro systems in combination with high-content image analysis (HCA) might contribute to DNT testing with lower costs, increased throughput and enhanced predictivity for human hazard identification. Human neural progenitor cells (hNPCs) grown as 3D neurospheres mimic basic processes of brain development including hNPC migration and differentiation and are therefore useful for DNT hazard identification. HCA of migrated neurospheres creates new challenges for automated evaluations because it encompasses variable cell densities, inconsistent z-layers and heterogeneous cell populations. We tackle those challenges with our Omnisphero software, which assesses multiple endpoints of the 'Neurosphere Assay.' For neuronal identification, Omnisphero reaches a true positive rate (TPR) of 83.8 % and a false discovery rate (FDR) of 11.4 %, thus being comparable to the interindividual difference among two researchers (TPR = 94.3, FDR = 11.0 %) and largely improving the results obtained by an existing HCA approach, whose TPR does not exceed 50 % at a FDR above 50 %. The high FDR of existing methods results in incorrect measurements of neuronal morphological features accompanied by an overestimation of compound effects. Omnisphero additionally includes novel algorithms to assess 'neurosphere-specific' endpoints like radial migration and neuronal density distribution within the migration area. Furthermore, a user-assisted parameter optimization procedure makes Omnisphero accessible to non-expert end users.

Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro.

Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the "Neurosphere Assay" to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to ß1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Automatic counting and positioning of 5-bromo-2-deoxyuridine (BrdU) positive cells in cortical layers of rat brain slices.

5-Bromo-2-deoxyuridine (BrdU) staining is often used to evaluate cortical layer formation during mammalian brain development. This method allows the quantification of newly generated cells and therefore the study of the effects of xenobiotics or genetic factors on proliferation, cell death and migration behavior in a quantitative manner. However, these endpoints are generally assessed by time-consuming manual evaluation. In the present work, we introduce a novel procedure to identify and quantify BrdU(+) cells within cortical layers, using the commercially available vHCS-Scan V.6.3.1 software to identify BrdU(+) cell coordinates and the novel program 'BrdeLuxe' to define cortical layers and quantitatively assign BrdU(+) cells to them. This procedure is compared to BrdU(+) cell counting with the freeware 'ImageJ' in respect to the manual evaluation, all by two different researchers. BrdeLuxe shows high accuracy and precision for the determination of total number of BrdU(+) cells compared to the manual counting, while ImageJ does not reach such results. Accuracy and precision are also higher for employing the BrdeLuxe program to evaluate the percentage of BrdU(+) cells per brain layer compared to ImageJ. In terms of running time, BrdeLuxe is the fastest method of the three making it more suitable for multiple brain slices analyses.

The Arg389Gly beta1-adrenoceptor polymorphism does not affect cardiac effects of exercise after parasympathetic inhibition by atropine.

UNLABELLED: In vitro, Arg389Gly beta1-adrenoceptor (AR) polymorphism exhibits decreased beta-AR signalling. In vivo, beta1-AR-mediated cardiac effects of exercise showed no genotype-dependent differences in Arg389 vs. Gly389 beta1-AR subjects. We studied in 16 male subjects homozygous Arg389 or Gly389 beta1-AR, whether blockade of parasympathetic activity might unmask genotype-dependence of exercise effects. Subjects were infused with atropine (10 microg/kg i.v. loading dose followed by continuous i.v. infusion of 0.15 microg/kg/min throughout exercise-time); 20 min after start of atropine bicycle-exercise in supine position (25, 50, 75 and 100 W for 5 min each) was performed and heart rate, contractility, blood pressure, plasma noradrenaline and plasma-renin activity were assessed. Exercise-evoked increases in all but one parameters were not different between Arg389 and Gly389 beta1-AR subjects; only plasma noradrenaline increased slightly more in Gly389 vs. Arg389 beta1-AR subjects. IN CONCLUSION: It appears to be unlikely that lack of Arg389Gly beta1-AR genotype-dependence of exercise-effects can be explained by influences of parasympathetic activity.

The Arg389Gly beta1-adrenoceptor polymorphism and catecholamine effects on plasma-renin activity.

OBJECTIVES: The purpose of this research was to find out whether, in humans, dobutamine-induced hemodynamic effects and increase in plasma-renin activity (PRA) might be beta1-adrenoceptor (beta1AR) genotype-dependent. BACKGROUND: In vitro Arg389Gly-beta1AR polymorphism exhibits decreased receptor signaling. METHODS: We studied 10 male homozygous Arg389-beta1AR subjects and 8 male homozygous Gly389beta1AR subjects; to avoid influences of codon 49 polymorphism, all were homozygous Ser49-beta1AR. Subjects were infused with dobutamine (1 to 6 microg/kg/min) with or without bisoprolol (10 mg orally) pretreatment, and PRA, heart rate, contractility, and blood pressure were assessed. RESULTS: With regard to PRA, dobutamine increased PRA more potently in Arg389-beta1AR versus Gly389-beta1AR subjects. Bisoprolol markedly suppressed the dobutamine-induced PRA increase in Arg389- but only marginally in Gly389-beta1AR subjects. With regard to hemodynamics, dobutamine caused larger heart rate and contractility increases and diastolic blood pressure decreases in Arg389- versus Gly389-beta1AR subjects. Bisoprolol reduced dobutamine-induced heart rate and contractility increases and diastolic blood pressure decreases more potently in Arg389- versus Gly389-beta1AR subjects. CONCLUSIONS: Codon 389 beta1AR polymorphism is a determinant not only of hemodynamic effects but also of PRA. Thus, beta1AR polymorphisms may be useful for predicting therapeutic responses to betaAR-blocker treatment.

Beta 2-adrenoceptor-mediated intrinsic sympathomimetic activity of carteolol: an in vivo study.

The intrinsic sympathomimetic activity (ISA) of a beta-adrenoceptor blocker can be mediated by beta(1)- or beta(2)-adrenoceptors. The aim of this study was to characterize the ISA of the beta-adrenoceptor blocker carteolol in healthy volunteers. Two approaches were employed. First, we assessed the effects of carteolol (20, 40 or 80 mg p.o.) on blood pressure, heart rate and heart-rate corrected duration of electromechanical systole (QS(2)c, a measure of cardiac contractility) in the volunteers. Carteolol dose-dependently increased systolic blood pressure, heart rate and contractility and decreased diastolic blood pressure. The beta(1)-adrenoceptor blocker bisoprolol did not attenuate these carteolol effects, but rather enhanced the effects on heart rate and systolic blood pressure. Second, we treated volunteers for 7 days with 1 x 20 mg/day carteolol and assessed lymphocyte beta(2)-adrenoceptor density (by (-)-[(125)I]-iodocyanopindolol binding) and functional responsiveness (by 10 muM isoprenaline-induced increase in lymphocyte cyclic AMP content). Carteolol significantly reduced lymphocyte beta(2)-adrenoceptor density and function. After withdrawal of carteolol lymphocyte beta(2)-adrenoceptor density and function recovered only very slowly and had not returned to control levels 11 days after carteolol withdrawal. In conclusion, the fact that, on the one hand, the cardiovascular effects of carteolol were not attenuated by the beta(1)-adrenoceptor blocker bisoprolol and, on the other, carteolol significantly decreased lymphocyte beta(2)-adrenoceptor density and function is in favour of the idea that the ISA of carteolol is mediated by beta(2)-adrenoceptors. Involvement of an additional receptor site (e.g. the propranolol-resistant state of the beta(1)-adrenoceptor), however, cannot be excluded.