Latest Articles
Research Article
July 12, 2018, updated July 17, 2018
Replacement of foot-and-mouth disease virus cattle tongue titration by in vitro titration
Aldo Dekker, Froukje van Hemert-Kluitenberg, Anna H. Oosterbaan, Kimberly Moonen, Laure Mouton
doi:10.14573/altex.1712222
Abstract
Titration of foot-and-mouth disease cattle challenge virus in cattle tongue has been the standard for many years in many countries, although for all other applications titration in animals has be replaced by in vitro methods. The objective of the analysis was the replacement of in vivo titration of cattle challenge virus by in vitro titration. Using data from 32 in vivo titration experiments together with the in vitro titration results, obtained by plaque count on primary lamb or pig kidney cells, of the same samples, as well as data from the virus isolation control chart used in the laboratory, we show that the repeatability of the in vitro titration is much higher than the repeatability of the in vivo titration. The titer on primary kidney cells was on average 1.4 log10 higher than the titer determined by titration in cattle tongue (PFU/ml compared to bovine ID50/ml), but the difference varied among different strains. The study also shows that the probability of infection in cattle tongue is high even when a lower challenge dose is used, which makes the variability between strains less important. Based on these results we propose to change the standard dose for cattle challenge from 104 bovine ID50 to 105.4 PFU, and therefore replace the in vivo cattle tongue titration method with the in vitro titration method.
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July 12, 2018
Review Article
Investigating cell type specific mechanisms contributing to acute oral toxicity
Pilar Prieto, Rabea Graepel, Kirsten Gerloff, Lara Lamon, Magdalini Sachana, Francesca Pistollato, Laura Gribaldo, Anna Bal-Price, and Andrew Worth
doi:10.14573/altex.1805181
Supplementary file 1
Supplementary file 2
Abstract
The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.
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July 8, 2018
Research Article
A human population-based organotypic in vitro model for cardiotoxicity screening
Fabian A. Grimm, Alexander Blanchette, John S. House, Kyle Ferguson, Nan-Hung Hsieh, Chimeddulam Dalaijamts, Alec A. Wright, Blake Anson, Fred A. Wright, Weihsueh A. Chiu and Ivan Rusyn
doi:10.14573/1805301
Abstract
Assessing inter-individual variability in responses to xenobiotics remains a substantial challenge, both in drug development with respect to pharmaceuticals and in public health with respect to environmental chemicals. Although approaches exist to characterize pharmacokinetic variability, there are no methods to routinely address pharmacodynamic variability. In this study, we aimed to demonstrate the feasibility of characterizing inter-individual variability in a human in vitro model. Specifically, we hypothesized that genetic variability across a population of iPSC-derived cardiomyocytes translates into reproducible variability in both baseline phenotypes and drug responses. We measured baseline and drug-related effects in iPSC-derived cardiomyocytes from 27 healthy donors on kinetic Ca2+ flux and high-content live cell imaging. Cells were treated in concentration-response with cardiotoxic drugs: isoproterenol (β-adrenergic receptor agonist/positive inotrope), propranolol (β-adrenergic receptor antagonist/negative inotrope), and cisapride (hERG channel inhibitor/QT prolongation). Cells from four of the 27 donors were further evaluated in terms of baseline and treatment-related gene expression. Reproducibility of phenotypic responses was evaluated across batches and time. iPSC-derived cardiomyocytes exhibited reproducible donor-specific differences in baseline function and drug-induced effects. We demonstrate the feasibility of using a panel of population-based organotypic cells from healthy donors as an animal replacement experimental model. This model can be used to rapidly screen drugs and chemicals for inter-individual variability in cardiotoxicity. This approach demonstrates the feasibility of quantifying inter-individual variability in xenobiotic responses and can be expanded to other cell types for which in vitro populations can be derived from iPSCs.
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July 8, 2018
Research Article
Brian D. Chapron, Alenka Chapron, Brian Phillips, Miracle C. Okoli, Danny D. Shen, Edward J. Kelly, Jonathan Himmelfarb and Kenneth E. Thummel
doi:10.14573/altex.1803161
Abstract
The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in the rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3–mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysiological systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.
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June 14, 2018
Review Article
Lindsey K. Borton and Kelly P. Coleman
doi:10.14573/altex.1709221
Abstract
Pyrogenicity presents a challenge to clinicians, medical device manufactures, and regulators. A febrile response may be caused by endotoxin contamination, microbial components other than endotoxin, or chemical agents that generate a material-mediated pyrogenic response. While test methods for the assessment of endotoxin contamination and some microbial components other than endotoxin are well-established, material-mediated pyrogens remain elusively undefined. This review presents the findings of literature searches conducted to identify material-mediated pyrogens associated with medical devices.
The in vivo rabbit pyrogen test (RPT) is considered to be the “gold standard” for medical device pyrogenicity testing, despite the fact that few medical device-derived material-mediated pyrogens are known. In line with global efforts to reduce the use of research animals, an in vitro monocyte activation test (MAT) has the potential to replace the RPT. The MAT is used to detect substances that activate human monocytes to release cytokines. This review will also describe the potential opportunities and challenges associated with MAT adoption for the detection of material-mediated pyrogens in medical device testing.
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June 13, 2018
Research Article
Ann-Cathrin Volz, Larissa Hack, Franziska B. Atzinger and Petra J. Kluger
doi:10.14573/altex.1802191
Abstract
Vascularized adipose tissue models are highly demanded as alternative to existing animal models to elucidate the mechanisms of widespread diseases, screen for new drugs or asses corresponding safety levels. Standardly used animal-derived sera therein, are associated to ethical concerns, the risk of contaminations and many uncertainties in their composition and impact on cells. Therefore, their use should be completely omitted. In this study, we developed a serum-free, defined co-culture medium and implemented it to set up an adipocyte/endothelial cell (EC) co-culture model.
Human adipose-derived stem cells were differentiated under defined conditions (diffASCs) and, like human microvascular ECs (mvECs), cultured in a developed defined co-culture medium in mono-, indirect or direct co-culture for 14 days. The developed defined co-culture medium was superior to compared mono-culture media and facilitated the functional maintenance and maturation of diffASCs including perilipin A expression, lipid accumulation and also glycerol and leptin release. The medium equally allowed mvEC maintenance, confirmed by the expression of cluster of differentiation 31 (CD31) and von Willebrand factor (vWF) and acetylated low-density lipoprotein (acLDL) uptake. Thereby mvECs showed a strong dependency on EC-specific factors. Additionally, the development of vascular structures by mvECs was facilitated when directly co-cultured with diffASCs.
The completely defined co-culture system allows for the serum-free setup of adipocyte/EC co-cultures and thereby represents a valuable and ethically acceptable tool for the setup of vascularized adipose tissue models.
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June 13, 2018
Research Article
Adaptation of the human Cell Line Activation Test (h-CLAT) to Animal-Product-Free Conditions
Alexander Edwards, Lottie Roscoe, Christopher Longmore, Fiona Bailey, Bushra Sim and Carol Treasure
doi:10.14573/altex.1710051
Abstract
Skin sensitisers are substances that can elicit allergic responses following skin contact and the process by which this occurs is described as skin sensitisation. Skin sensitisation is defined as a series of key events, that form an adverse outcome pathway (AOP). Key event three in the AOP is dendritic cell activation that can be modelled by the human Cell Line Activation Test (h-CLAT) and is typified by changes in cell surface markers CD54 and CD86 in dendritic cells. The h-CLAT is accepted at a regulatory level (OECD Test-Guideline (TG)442E) and can be used to assess skin sensitisation potential as part of an integrated approach to testing and assessment (IATA).
Stakeholders in the cosmetics and chemical industries have scientific and ethical concerns relating to use of animal derived material and have communicated a strong preference for fully human based in vitro methods. Therefore, we adapted the h-CLAT to animal-product-free conditions and validated the adapted method with the proficiency panel substances in Annex II of TG442E, using 3 independent batches of pooled human serum. The modified method showed equivalence to the validated reference method (VRM), as all proficiency substances were correctly classified. Comparable values for CV75 (concentration yielding 75% cell viability), EC150 and EC200 (concentration yielding RFI of ≥150 for CD86 and ≥200 for CD54) were obtained. Data generated using the adapted method may be used in European REACH submissions, provided the proficiency data is included. We are seeking formal inclusion of the adaptation into TG442E, enabling compliance with global regulations.
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March 11, 2018
Research Article
Testing vaginal irritation with the hen’s egg test-chorioallantoic membrane assay
Rita Palmeira-de-Oliveira, Rita Monteiro Machado, José Martinez-de-Oliveira and Ana Palmeira-de-Oliveira
doi : 10.14573/altex.1710091
Abstract
The HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) assay is an in vitro alternative to the in vivo Draize Rabbit Eye test that mimics vascular changes in the chorioallantoic membrane. This qualitative method assesses the irritancy potential of chemicals. The CAM responds to injury with an inflammatory process similar to that in the rabbit eye’s conjunctival tissue. Regarding topical toxicity assessment of medical devices, ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irritation potential for the eye and the vaginal epithelia is similar. The aim of this work was to apply the HET-CAM assay to test the irritancy potential of vaginal formulations. Vaginal semisolid medicines and lubricants currently marketed were tested along with the Universal Placebo formulation that has been shown to be clinically safe. Nonoxynol-9 (N-9), a known vaginal irritant, was enrolled as positive control (concentrations ranging from 0.001 to 100% (v/v)). The assay was conducted according to the ICCVAM - Recommended test method (NIH Publication No. 10-7553 – 2010). Formulations were then classified according to irritation score (IS), using the analysis methods (A) and (B). The studied vaginal formulations showed low potential for irritation. N-9 was classified as a severe irritant at concentrations above 2%, which corroborates clinical data, envisaging a possible in vitro/in vivo correlation. IS (B) was considered a better classification output. Although still requiring further validation, the HET-CAM assay seems an ideal prospect for vaginal irritancy potential in vitro studies.