Food for thought ... considerations and guidelines for basic test method descriptions in toxicology

The development and application of new test systems and test methods is central to the progress of in vitro toxicology. In order to live up to the future challenges, it is important to use the vast knowledge of adjoining fields, such as cell biology or developmental biology, and to attract specialists from such areas to develop new methods. Not all of them bring with them the necessary experience and training required for the development of toxicological test systems. Thus, promising new biological test systems sometimes still require additional considerations to become successful. Sometimes even the referees of scientific journals and their editors seem to lack judgement guidelines for minimum performance standards. Here we provide a list of points to be considered for the establishment of a test method. The chapters range from the explanation of the meaning of positive controls, performance standards or signal-noise ratios to a discussion of statistical considerations, suitable solvents and data display formats. The considerations are simple and expressed in a non-mathematical format, with a strong focus on plausibility and common sense. The major intention of this article is to provide a compilation of important issues requiring consideration. Whether they apply to a specific system and whether action is required must be determined by individual judgement

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

Astrocytes, the largest cell population in the human brain, are powerful inflammatory effectors. Several studies have examined the interaction of activated astrocytes with neurons, but little is known yet about human neurotoxicity under such situations and about strategies of neuronal rescue. To address this question, immortalized murine astrocytes (IMA) were combined with human LUHMES neurons and stimulated with an inflammatory (TNF, IL-1) cytokine mix (CM). Neurotoxicity was studied both in co-cultures and in monocultures after transfer of conditioned medium from activated IMA. Interventions with >20 drugs were used to profile the model system. Control IMA supported neurons and protected them from neurotoxicants. Inflammatory activation reduced this protection, and prolonged exposure of co-cultures to CM triggered neurotoxicity. Neither the added cytokines nor the release of NO from astrocytes were involved in this neurodegeneration. The neurotoxicity-mediating effect of IMA was faithfully reproduced by human astrocytes. Moreover, glia-dependent toxicity was also observed, when IMA cultures were stimulated with CM, and the culture medium was transferred to neurons. Such neurotoxicity was prevented when astrocytes were treated by p38 kinase inhibitors or dexamethasone, whereas such compounds had no effect when added to neurons. Conversely, treatment of neurons with five different drugs, including resveratrol and CEP1347, prevented toxicity of astrocyte supernatants. Thus, the sequential IMA-LUHMES neuroinflammation model is suitable for separate profiling of both glial-directed and directly neuroprotective strategies. Moreover, direct evaluation in co-cultures of the same cells allows for testing of therapeutic effectiveness in more complex settings, in which astrocytes affect pharmacological properties of neurons.publishe

Prevention of the degeneration of human dopaminergic neurons in an astrocyte co-culture system allowing endogenous drug metabolism

Background and purpose Few neuropharmacological model systems use human neurons. Moreover, available test systems rarely reflect functional roles of co-cultured glial cells. There is no human in vitro counterpart of the widely used 1-methyl-4-phenyl-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Experimental Approach We generated such a model by growing an intricate network of human dopaminergic neurons on a dense layer of astrocytes. In these co-cultures, MPTP was metabolized to 1-methyl-4-phenyl-pyridinium (MPP+) by the glial cells, and the toxic metabolite was taken up through the dopamine transporter into neurons. Cell viability was measured biochemically and by quantitative neurite imaging, siRNA techniques were also used. Key Results We initially characterized the activation of PARP. As in mouse models, MPTP exposure induced (poly-ADP-ribose) synthesis and neurodegeneration was blocked by PARP inhibitors. Several different putative neuroprotectants were then compared in mono-cultures and co-cultures. Rho kinase inhibitors worked in both models; CEP1347, ascorbic acid or a caspase inhibitor protected mono-cultures from MPP+ toxicity, but did not protect co-cultures, when used alone or in combination. Application of GSSG prevented degeneration in co-cultures, but not in mono-cultures. The surprisingly different pharmacological profiles of the models suggest that the presence of glial cells, and the in situ generation of the toxic metabolite MPP+ within the layered cultures played an important role in neuroprotection.Conclusions and Implications Our new model system is a closer model of human brain tissue than conventional cultures. Its use for screening of candidate neuroprotectants may increase the predictiveness of a test battery

Characterization of mouse cell line IMA 2.1 as a potential model system to study astrocyte functions

Astrocytes are activated in most chronic neurodegenerative diseases associated with inflammatory events such as Parkinson’s disease or Alzheimer’s disease, but also in stroke. Due to an aging population worldwide, research efforts in these areas are likely to expand in the future. This will entail an increaseddemand for appropriate experimental models. We introduce here the new immortalized mouse astrocyte cell line IMA 2.1 as an alternative to currently used primary astrocyte cultures. IMA 2.1 were directly compared with primary mouse astrocytes with respect to their response to proinflammatory stimuli, expression of typical astrocyte markers, and to the cell line’s capacity to metabolize the parkinsonian toxin MPTP to its toxic metabolite MPP+. Under inflammatory conditions, mimicked with the addition of a cytokine mix, IMA 2.1 responded similarly to primary astrocytes with mRNA upregulation, expression of iNOS and COX-2, and the release of various inflammatory mediators. Analysis of astrocytic markers indicated that IMA 2.1 represent a relatively early, GFAP-negative stage of astrocyte development. Moreover, conversion of MPTP by monoamine oxidase-B proceeded in IMA at least as quickly as in primary cells. For all endpoints investigated, the cell line IMA 2.1, derived from a single clone, delivered reproducible results over a period of several years and allowed upscaling of experiments due to its easy handling compared with primary cells

Neurothreads: Development of supportive carriers for mature dopaminergic neuron differentiation and implantation

In this study we present the use of elastic macroporous cryogels for differentiation and transplantation of mature neurons. We develop a coating suitable for long-term neuronal culture, including stem cell differentiation, by covalent immobilization of neural adhesion proteins. In the context of cell therapy for Parkinson's disease, we show compatibility with established dopaminergic differentiation of both immortalized mesencephalic progenitors - LUHMES - and human embryonic stem cells (hESCs). We adjust structural properties of the biomaterial to create carriers - Neurothreads - favourable for cell viability during transplantation. Finally, we show feasibility of preservation of mature neurons, supported by Neurothreads, one month after in-vivo transplantation. Preliminary data suggests that the Neurothread approach could provide more mature and less proliferative cells in vivo

Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift

Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored, little is known about effects of minor, possibly pleiotropic, genome changes. We addressed this question for the human dopaminergic neuronal precursor cell line LUHMES by comparing two subpopulations (SP) maintained either at the American-Type-Culture-Collection (ATCC) or by the original provider (UKN). Drastic differences in susceptibility towards the specific dopaminergic toxicant 1-methyl-4-phenylpyridinium (MPP+) were observed. Whole-genome sequencing was performed to identify underlying genetic differences. While both SP had normal chromosome structures, they displayed about 70 differences on the level of amino acid changing events. Some of these differences were confirmed biochemically, but none offered a direct explanation for the altered toxicant sensitivity pattern. As second approach, markers known to be relevant for the intended use of the cells were specifically tested. The "ATCC" cells rapidly down-regulated the dopamine-transporter and tyrosine-hydroxylase after differentiation, while "UKN" cells maintained functional levels. As the respective genes were not altered themselves, we conclude that polygenic complex upstream changes can have drastic effects on biochemical features and toxicological responses of relatively similar SP of cells.publishe

Generation of Genetically-Modified Human Differentiated Cells for Toxicological Tests and the Study of Neurodegenerative Diseases

Human differentiated cell types, such as neurons or hepatocytes, are of limited availability, and their use for experiments requiring ectopic gene expression is challenging. Using the human conditionally-immortalized neuronal precursor line LUHMES, we explored whether genetic modification in the proliferating state could be used for experiments in the differentiated post-mitotic neurons. First, alpha-synuclein (ASYN), a gene associated with the pathology of Parkinson’s disease, was overexpressed. Increased amounts of the protein were tolerated without change of phenotype, and this approach now allows further studies on protein variants. Knockdown of ASYN attenuated the toxicity of the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP+). Different lentiviral constructs then were tested: cells labeled ubiquitously with green (GFP) or red fluorescent protein (RFP) allowed the quantification of neurite growth and of its disturbance by toxicants; expression of proteins of interest could be targeted to different organelles; production of two different proteins from a single read-through construct was achieved successfully by an expression strategy using a linker peptide between the two proteins, which is cleaved by deubiquitinases; LUHMES, labeled with GFP in the cytosol and RFP in the mitochondria, were used to quantify mitochondrial mobility along the neurites. MPP+ reduced such organelle movement before any other detectable cellular change, and this toxicity was prevented by simultaneous treatment with the antioxidant ascorbic acid. Thus, a strategy has been outlined here to study new functional endpoints, and subtle changes of structure and proteostasis relevant in toxicology and biomedicine in post-mitotic human cells

Determination of Risk Factors for Severe Life-Threatening Course of Multisystem Inflammatory Syndrome Associated with COVID-19 in Children

Multisystem inflammatory syndrome associated with COVID-19 in children (MIS-C) is a life-threatening condition that often requires intensive care unit (ICU) admission. The aim of this study was to determine risk factors for severe/life-threatening course of MIS-C. The study included 166 patients (99 boys, 67 girls) aged 4 months–17 years (median 8.2 years). The criterion of severity was the fact of ICU admission. To conduct a comparative analysis, MIS-C patients were divided into two groups: patients hospitalized in the ICU (n = 84, 50.6%) and those who did not need ICU admission (n = 82, 49.4%). Patients with a more severe course of MIS-C were significantly older. They had a higher frequency of signs such as rash, swelling, hepatomegaly, splenomegaly, and neurological and respiratory symptoms. Hypotension/shock and myocardial involvement were much more common in patients with severe MIS-C. These patients had a more significant increase in CRP, creatinine, troponin, and D-dimer levels. Additionally, the presence of macrophage activation syndrome was higher in patients admitted to the ICU. Conclusion: Nineteen predictors of severe course of MIS-C were found, out of which hepatomegaly, splenomegaly, D-dimer > 2568 ng/mL, troponin > 10 pg/mL were mainly associated with the probability of being classified as early predictors of severe MIS-C requiring ICU admission