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The first ONTOX Hackathon of the EU Horizon 2020-funded ONTOX project was held on 21-23 April 2024 in Utrecht, The Netherlands (https://ontox-project.eu/hackathon/). This participatory event aimed to collectively advance innovation for human safety through the use of Artificial Intelligence (AI), and hence significantly reduce reliance on animal-based testing. Expert scientists, industry leaders, young investigators, members of animal welfare organisations and academics alike, joined the hackathon. Eight teams were stimulated to find innovative solutions for challenging themes, that were selected based on previous discussions between stakeholders, namely: How to drive the use of AI in chemical risk assessment?; To predict or protect?; How can we secure human health and environmental protection at the same time?; and How can we facilitate the transition from animal tests to full implementation of human-relevant methods? The hackathon ended with a pitching contest, where the teams presented their solutions to a jury. The most promising solutions will be presented to regulatory authorities, industry, academia and non-governmental organisations at the next ONTOX Stakeholder Network meeting and taken up by the ONTOX project in order to tackle the above-mentioned challenges further. This report comprises two parts: The first part highlights some of the lessons learnt during the planning and execution of the hackathon; the second part presents the outcome of the ONTOX Hackathon, which resulted in several innovative and promising solutions based on New Approach Methodologies (NAMs), and outlines ONTOX's intended way forward., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The Virtual Human Platform for Safety Assessment (VHP4Safety) project aims to build a Virtual Human Platform (VHP) to protect human health and revolutionize the safety assessment of chemicals and pharmaceuticals by transitioning from animal-based to human-based approaches. The goal of this article is to introduce the project and its interdisciplinary approach to co-creation with multiple academic, regulatory, industrial and societal partners covering the entire safety assessment knowledge chain. Three research lines drive the project: 1) building the VHP; 2) feeding the VHP with human data; and 3) implementing the VHP. The project focusses on three case studies that incorporate human relevant scenarios not included in current animal-based safety assessment strategies. The VHP is built on tools and services, including pharmacokinetic and computational models, and integrates several data sources within each case study, including data on human physiology, epidemiology, toxicokinetic and -dynamic parameters, as well as data on chemical characteristics and exposures. In addition, the VHP integrates new data generated within the project using new approach methodologies representing key events within adverse outcome pathways. Implementation of the VHP is investigated using an innovation systems approach, engaging stakeholders and organizing training and education. Central to the VHP4Safety project is our co-creative approach, which facilitated by biannual designathons and hackathons that foster active involvement of all project participants from over 30 partner organizations. By integrating technological innovations with transparency and stakeholder collaboration, the VHP4Safety project will help shape the transition to the next generation safety assessment in which animal testing becomes redundant.

Researchers in biomedical research, public health and the life sciences often spend weeks or months discovering, accessing, curating, and integrating data from disparate sources, significantly delaying the onset of actual analysis and innovation. Instead of countless developers creating redundant and inconsistent data pipelines, BioBricks.ai offers a centralized data repository and a suite of developer-friendly tools to simplify access to scientific data. Currently, BioBricks.ai delivers over ninety biological and chemical datasets. It provides a package manager-like system for installing and managing dependencies on data sources. Each 'brick' is a Data Version Control git repository that supports an updateable pipeline for extraction, transformation, and loading data into the BioBricks.ai backend at https://biobricks.ai. Use cases include accelerating data science workflows and facilitating the creation of novel data assets by integrating multiple datasets into unified, harmonized resources. In conclusion, BioBricks.ai offers an opportunity to accelerate access and use of public data through a single open platform., Competing Interests: 10.Conflict of Interest The authors declare the following potential conflicts of interest regarding the research and publication of this paper: BioBricks is a product developed by Insilica LLC, and many of the authors are employees of Insilica LLC. As such, there may be a perceived or real financial interest in the outcomes of the research and the development of BioBricks. The authors affirm that their contributions to the research and the manuscript were conducted with scientific integrity and without bias influenced by their association with Insilica LLC.

Objective: To develop and internally validate a prognostic model to predict chronic pain after a new episode of acute or subacute non-specific idiopathic, non-traumatic neck pain in patients presenting to physiotherapy primary care, emphasising modifiable biomedical, psychological and social factors., Design: A prospective cohort study with a 6-month follow-up between January 2020 and March 2023., Setting: 30 physiotherapy primary care practices., Participants: Patients with a new presentation of non-specific idiopathic, non-traumatic neck pain, with a duration lasting no longer than 12 weeks from onset., Baseline Measures: Candidate prognostic variables collected from participants included age and sex, neck pain symptoms, work-related factors, general factors, psychological and behavioural factors and the remaining factors: therapeutic relation and healthcare provider attitude., Outcome Measures: Pain intensity at 6 weeks, 3 months and 6 months on a Numeric Pain Rating Scale (NPRS) after inclusion. An NPRS score of ≥3 at each time point was used to define chronic neck pain., Results: 62 (10%) of the 603 participants developed chronic neck pain. The prognostic factors in the final model were sex, pain intensity, reported pain in different body regions, headache since and before the neck pain, posture during work, employment status, illness beliefs about pain identity and recovery, treatment beliefs, distress and self-efficacy. The model demonstrated an optimism-corrected area under the curve of 0.83 and a corrected R 2 of 0.24. Calibration was deemed acceptable to good, as indicated by the calibration curve. The Hosmer-Lemeshow test yielded a p-value of 0.7167, indicating a good model fit., Conclusion: This model has the potential to obtain a valid prognosis for developing chronic pain after a new episode of acute and subacute non-specific idiopathic, non-traumatic neck pain. It includes mostly potentially modifiable factors for physiotherapy practice. External validation of this model is recommended., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The field of chemical toxicity testing is undergoing a transition to overcome the limitations of in vivo experiments. This evolution involves implementing innovative non-animal approaches to improve predictability and provide a more precise understanding of toxicity mechanisms. Adverse outcome pathway (AOP) networks are pivotal in organizing existing mechanistic knowledge related to toxicological processes. However, these AOP networks are dynamic and require regular updates to incorporate the latest data. Regulatory challenges also persist due to concerns about the reliability of the information they offer. This study introduces a generic Weight-of-Evidence (WoE) scoring method, aligned with the tailored Bradford-Hill criteria, to quantitatively assess the confidence levels in key event relationships (KERs) within AOP networks. We use the previously published AOP network on chemical-induced liver steatosis, a prevalent form of human liver injury, as a case study. Initially, the existing AOP network is optimized with the latest scientific information extracted from PubMed using the free SysRev platform for artificial intelligence (AI)-based abstract inclusion and standardized data collection. The resulting optimized AOP network, constructed using Cytoscape, visually represents confidence levels through node size (key event, KE) and edge thickness (KERs). Additionally, a Shiny application is developed to facilitate user interaction with the dataset, promoting future updates. Our analysis of 173 research papers yielded 100 unique KEs and 221 KERs among which 72 KEs and 170 KERs, respectively, have not been previously documented in the prior AOP network or AOP-wiki. Notably, modifications in de novo lipogenesis, fatty acid uptake and mitochondrial beta-oxidation, leading to lipid accumulation and liver steatosis, garnered the highest KER confidence scores. In conclusion, our study delivers a generic methodology for developing and assessing AOP networks. The quantitative WoE scoring method facilitates in determining the level of support for KERs within the optimized AOP network, offering valuable insights into its utility in both scientific research and regulatory contexts. KERs supported by robust evidence represent promising candidates for inclusion in an in vitro test battery for reliably predicting chemical-induced liver steatosis within regulatory frameworks., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mathieu Vinken reports financial support was provided by the Horizon2020 Research and Innovation Framework program. Mathieu Vinken reports financial support was provided by the Flemish government (Methusalem program). Mathieu Vinken reports financial support was provided by the Research Foundation Flanders. Mathieu Vinken reports financial support was provided by the Scientific Fund Willy Gepts. Mathieu Vinken reports financial support was provided by the Center for Alternatives to Animal Testing. Mathieu Vinken reports financial support was provided by the Alternatives Research and Development Foundation. Tamara Vanhaecke reports financial support was provided by the Colgate-Palmolive Grant (Society of Toxicology). Tamara Vanhaecke reports financial support was provided by Onderzoeksraad Vrije Universiteit Brussel. Tamara Vanhaecke reports financial support was provided by Brussels Environment of the Brussels-Capital Region. Tamara Vanhaecke reports financial support was provided by the Research Chair Mireille Aerens for the development of Alternatives to Animal Testing. Mathieu Vinken, editor-in-chief of Toxicology, acts as co-author of this submission. He will not be involved in the handling and evaluation of the manuscript. Furthermore, this submission will be blinded for Mathieu Vinken in the online Editorial Manager system in order to avoid conflict of interest issues If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To study the ways in which compounds can induce adverse effects, toxicologists have been constructing Adverse Outcome Pathways (AOPs). An AOP can be considered as a pragmatic tool to capture and visualize mechanisms underlying different types of toxicity inflicted by any kind of stressor, and describes the interactions between key entities that lead to the adverse outcome on multiple biological levels of organization. The construction or optimization of an AOP is a labor intensive process, which currently depends on the manual search, collection, reviewing and synthesis of available scientific literature. This process could however be largely facilitated using Natural Language Processing (NLP) to extract information contained in scientific literature in a systematic, objective, and rapid manner that would lead to greater accuracy and reproducibility. This would support researchers to invest their expertise in the substantive assessment of the AOPs by replacing the time spent on evidence gathering by a critical review of the data extracted by NLP. As case examples, we selected two frequent adversities observed in the liver: namely, cholestasis and steatosis denoting accumulation of bile and lipid, respectively. We used deep learning language models to recognize entities of interest in text and establish causal relationships between them. We demonstrate how an NLP pipeline combining Named Entity Recognition and a simple rules-based relationship extraction model helps screen compounds related to liver adversities in the literature, but also extract mechanistic information for how such adversities develop, from the molecular to the organismal level. Finally, we provide some perspectives opened by the recent progress in Large Language Models and how these could be used in the future. We propose this work brings two main contributions: 1) a proof-of-concept that NLP can support the extraction of information from text for modern toxicology and 2) a template open-source model for recognition of toxicological entities and extraction of their relationships. All resources are openly accessible via GitHub (https://github.com/ontox-project/en-tox)., Competing Interests: Author TL was employed by ToxTrack. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Corradi, Luechtefeld, de Haan, Pieters, Freedman, Vanhaecke, Vinken and Teunis.)

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Both because of the shortcomings of existing risk assessment methodologies, as well as newly available tools to predict hazard and risk with machine learning approaches, there has been an emerging emphasis on probabilistic risk assessment. Increasingly sophisticated AI models can be applied to a plethora of exposure and hazard data to obtain not only predictions for particular endpoints but also to estimate the uncertainty of the risk assessment outcome. This provides the basis for a shift from deterministic to more probabilistic approaches but comes at the cost of an increased complexity of the process as it requires more resources and human expertise. There are still challenges to overcome before a probabilistic paradigm is fully embraced by regulators. Based on an earlier white paper (Maertens et al., 2022), a workshop discussed the prospects, challenges and path forward for implementing such AI-based probabilistic hazard assessment. Moving forward, we will see the transition from categorized into probabilistic and dose-dependent hazard outcomes, the application of internal thresholds of toxicological concern for data-poor substances, the acknowledgement of user-friendly open-source software, a rise in the expertise of toxicologists required to understand and interpret artificial intelligence models, and the honest communication of uncertainty in risk assessment to the public.

Aim: To investigate the effects of exercise on salivary concentrations of inflammatory markers by analyzing a panel of 25 inflammatory markers in subjects who had participated in bicycle ergometer tests varying in workload and hydration status., Methods: Fifteen healthy young men (20-35 years) had performed 4 different exercise protocols of 1 hour duration in a randomly assigned cross-over design, preceded by a rest protocol. Individual workloads depended on participant's pre-assessed individual maximum workload (Wmax): rest (protocol 1), 70% Wmax in hydrated (protocol 2) and dehydrated (protocol 3) state, 50% Wmax (protocol 4) and intermittent 85%/55% Wmax in 2 min blocks (protocol 5). Saliva samples were collected before (T0) and immediately after exercise (T1), and at several time points after exercise (2 hours (T3), 3 hours (T4), 6 hours (T5) and 24 hours (T6)). Secretory Leukocyte Protease Inhibitor (SLPI), Matrix Metallopeptidase-9 (MMP-9) and lactoferrin was analyzed using a commercial ELISA kit, a panel of 22 cytokines and chemokines were analyzed using a commercial multiplex immunoassay. Data was analyzed using a multilevel mixed linear model, with multiple test correction., Results: Among a panel of 25 inflammatory markers, SLPI concentrations were significantly elevated immediately after exercise in all protocols compared to rest and higher concentrations reflected the intensity of exercise and hydration status. MMP-9 showed a significant increase in the 70% Wmax dehydrated, 50% Wmax and intermittent protocols., Conclusions: Salivary concentrations of SLPI and MMP-9 seem associated with exercise intensity and hydration status and may offer non-invasive biomarkers to study (local) inflammatory responses to different exercise intensities in human studies., Competing Interests: Karen Knipping, Nicole Buurman and Johan Garssen are employee of Danone Nutricia Research. All other authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Knipping et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Adverse outcome pathway (AOP) networks are versatile tools in toxicology and risk assessment that capture and visualize mechanisms driving toxicity originating from various data sources. They share a common structure consisting of a set of molecular initiating events and key events, connected by key event relationships, leading to the actual adverse outcome. AOP networks are to be considered living documents that should be frequently updated by feeding in new data. Such iterative optimization exercises are typically done manually, which not only is a time-consuming effort, but also bears the risk of overlooking critical data. The present study introduces a novel approach for AOP network optimization of a previously published AOP network on chemical-induced cholestasis using artificial intelligence to facilitate automated data collection followed by subsequent quantitative confidence assessment of molecular initiating events, key events, and key event relationships., Methods: Artificial intelligence-assisted data collection was performed by means of the free web platform Sysrev. Confidence levels of the tailored Bradford-Hill criteria were quantified for the purpose of weight-of-evidence assessment of the optimized AOP network. Scores were calculated for biological plausibility, empirical evidence, and essentiality, and were integrated into a total key event relationship confidence value. The optimized AOP network was visualized using Cytoscape with the node size representing the incidence of the key event and the edge size indicating the total confidence in the key event relationship., Results: This resulted in the identification of 38 and 135 unique key events and key event relationships, respectively. Transporter changes was the key event with the highest incidence, and formed the most confident key event relationship with the adverse outcome, cholestasis. Other important key events present in the AOP network include: nuclear receptor changes, intracellular bile acid accumulation, bile acid synthesis changes, oxidative stress, inflammation and apoptosis., Conclusions: This process led to the creation of an extensively informative AOP network focused on chemical-induced cholestasis. This optimized AOP network may serve as a mechanistic compass for the development of a battery of in vitro assays to reliably predict chemical-induced cholestatic injury., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mathieu Vinken reports financial support was provided by European Commission. Mathieu Vinken reports financial support was provided by Flemish government. Mathieu Vinken reports financial support was provided by Research Foundation Flanders. Mathieu Vinken reports financial support was provided by Scientific Fund Willy Gepts. Mathieu Vinken reports financial support was provided by Center for Alternatives to Animal Testing. Mathieu Vinken reports financial support was provided by Alternatives Research and Development Foundation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary: Xpaths is a collection of algorithms that allow for the prediction of compound-induced molecular mechanisms of action by integrating phenotypic endpoints of different species; and proposes follow-up tests for model organisms to validate these pathway predictions. The Xpaths algorithms are applied to predict developmental and reproductive toxicity (DART) and implemented into an in silico platform, called DARTpaths., Availability and Implementation: All code is available on GitHub https://github.com/Xpaths/dartpaths-app under Apache license 2.0, detailed overview with demo is available at https://www.vivaltes.com/dartpaths/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

Adverse Outcome Pathways (AOPs) are conceptual frameworks that tie an initial perturbation (molecular initiating event) to a phenotypic toxicological manifestation (adverse outcome), through a series of steps (key events). They provide therefore a standardized way to map and organize toxicological mechanistic information. As such, AOPs inform on key events underlying toxicity, thus supporting the development of New Approach Methodologies (NAMs), which aim to reduce the use of animal testing for toxicology purposes. However, the establishment of a novel AOP relies on the gathering of multiple streams of evidence and information, from available literature to knowledge databases. Often, this information is in the form of free text, also called unstructured text, which is not immediately digestible by a computer. This information is thus both tedious and increasingly time-consuming to process manually with the growing volume of data available. The advancement of machine learning provides alternative solutions to this challenge. To extract and organize information from relevant sources, it seems valuable to employ deep learning Natural Language Processing techniques. We review here some of the recent progress in the NLP field, and show how these techniques have already demonstrated value in the biomedical and toxicology areas. We also propose an approach to efficiently and reliably extract and combine relevant toxicological information from text. This data can be used to map underlying mechanisms that lead to toxicological effects and start building quantitative models, in particular AOPs, ultimately allowing animal-free human-based hazard and risk assessment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Implementation of reliable methodologies allowing Reduction, Refinement, and Replacement (3Rs) of animal testing is a process that takes several decades and is still not complete. Reliable methods are essential for regulatory hazard assessment of chemicals where differences in test protocol can influence the test outcomes and thus affect the confidence in the predictive value of the organisms used as an alternative for mammals. Although test guidelines are common for mammalian studies, they are scarce for non-vertebrate organisms that would allow for the 3Rs of animal testing. Here, we present a set of 30 reporting criteria as the basis for such a guideline for Developmental and Reproductive Toxicology (DART) testing in the nematode Caenorhabditis elegans . Small organisms like C. elegans are upcoming in new approach methodologies for hazard assessment; thus, reliable and robust test protocols are urgently needed. A literature assessment of the fulfilment of the reporting criteria demonstrates that although studies describe methodological details, essential information such as compound purity and lot/batch number or type of container is often not reported. The formulated set of reporting criteria for C. elegans testing can be used by (i) researchers to describe essential experimental details (ii) data scientists that aggregate information to assess data quality and include data in aggregated databases (iii) regulators to assess study data for inclusion in regulatory hazard assessment of chemicals., (Published by Oxford University Press 2021.)

Precision-cut intestinal slices (PCIS) are used to study intestinal (patho)physiology, drug efficacy, toxicity, transport and metabolism ex vivo. One of the factors that limit the use of PCIS is a relatively short life-span. Moreover, culture-induced changes in cellular composition of PCIS remain largely uncharacterized. In this study, we demonstrated the epithelial cell heterogeneity in mouse and rat PCIS and its alterations during culture. In addition, we evaluated whether the presence of niche growth factors impacts the survival of PCIS epithelial cells. We showed that freshly prepared PCIS retained the main epithelial cell types, namely absorptive enterocytes, goblet cells, enteroendocrine cells, stem cells, transit-amplifying cells and Paneth cells. Once placed in culture, PCIS displayed progressive epithelial damage, and loss of these epithelial cell types. Cells comprising the intestinal stem cell niche were especially sensitive to the damage, and the addition of niche growth factors beneficially affected the survival of stem cells and transit-amplifying cells in PCIS during culture. In conclusion, this study provides new insights into the dynamic changes in cellular composition of epithelium in cultured PCIS, paving the way to future toxicological and pharmacological studies in an informed and reliable ex vivo setting., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Strenuous physical stress induces a range of physiological responses, the extent depending, among others, on the nature and severity of the exercise, a person's training level and overall physical resilience. This principle can also be used in an experimental set-up by measuring time-dependent changes in biomarkers for physiological processes. In a previous report, we described the effects of workload delivered on a bicycle ergometer on intestinal functionality. As a follow-up, we here describe an analysis of the kinetics of various other biomarkers., Aim: To analyse the time-dependent changes of 34 markers for different metabolic and immunological processes, comparing four different exercise protocols and a rest protocol., Methods: After determining individual maximum workloads, 15 healthy male participants (20-35 years) started with a rest protocol and subsequently performed (in a cross-over design with 1-week wash-out) four exercise protocols of 1-h duration at different intensities: 70% W max in a hydrated and a mildly dehydrated state, 50% W max and intermittent 85/55% W max in blocks of 2 min. Perceived exertion was monitored using the Borg' Rating of Perceived Exertion scale. Blood samples were collected both before and during exercise, and at various timepoints up to 24 h afterward. Data was analyzed using a multilevel mixed linear model with multiple test correction., Results: Kinetic changes of various biomarkers were exercise-intensity-dependent. Biomarkers included parameters indicative of metabolic activity (e.g., creatinine, bicarbonate), immunological and hematological functionality (e.g., leukocytes, hemoglobin) and intestinal physiology (citrulline, intestinal fatty acid-binding protein, and zonulin). In general, responses to high intensity exercise of 70% W max and intermittent exercise i.e., 55/85% W max were more pronounced compared to exercise at 50% W max ., Conclusion: High (70 and 55/85% W max ) and moderate (50% W max ) intensity exercise in a bicycle ergometer test produce different time-dependent changes in a broad range of parameters indicative of metabolic activity, immunological and hematological functionality and intestinal physiology. These parameters may be considered biomarkers of homeostatic resilience. Mild dehydration intensifies these time-related changes. Moderate intensity exercise of 50% W max shows sufficient physiological and immunological responses and can be employed to test the health condition of less fit individuals., (Copyright © 2020 Kartaram, van Norren, Schoen, Teunis, Mensink, Verschuren, M’Rabet, Besseling-van der Vaart, Mohrmann, Wittink, Garssen, Witkamp and Pieters.)

Background & Aims: Plasma citrulline concentration is considered to be a marker for enterocyte metabolic mass and to reflect its reduction as may occur during intestinal dysfunction. Strenuous exercise can act as a stressor to induce small intestinal injury. Our previous studies suggest that this comprises the intestinal ability to produce citrulline from a glutamine-rich protein bolus. In this study we investigated the effects of different exercise intensities and hydration state on citrulline and iFABP levels following a post-exercise glutamine bolus in healthy young men., Methods: Fifteen healthy young men (20-35 yrs, VO 2 max 56.9 ± 3.9 ml kg -1  min -1 ) performed in a randomly assigned cross-over design, a rest (protocol 1) and four cycle ergometer protocols. The volunteers cycled submaximal at different percentages of their individual pre-assessed maximum workload (Wmax): 70% Wmax in hydrated (protocol 2) and dehydrated state (protocol 3), 50% Wmax (protocol 4) and intermittent 85/55% Wmax in blocks of 2 min (protocol 5). Immediately after 1 h exercise or rest, subjects were given a glutamine bolus with added alanine as an iso-caloric internal standard (7.5 g of each amino acid). Blood samples were collected before, during and after rest or exercise, up to 24 h post onset of the experiment. Amino acids and urea were analysed as metabolic markers, creatine phosphokinase and iFABP as markers of muscle and intestinal damage, respectively. Data were analysed using a multilevel mixed linear statistical model. p values were corrected for multiple testing., Results: Citrulline levels already increased before glutamine supplementation during normal hydrated exercise, while this was not observed in the dehydrated and rest protocols. The low intensity exercise protocol (50% Wmax) showed the highest increase in citrulline levels both during exercise (43.83 μmol/L ± 2.63 (p < 0.001)) and after glutamine consumption (50.54 μmol/L ± 2.62) compared to the rest protocol (28.97 μmol/L ± 1.503 and 41.65 μmol/L ± 1.96, respectively, p < 0.05). However, following strenuous exercise at 70% Wmax in the dehydrated state, citrulline levels did not increase during exercise and less after the glutamine consumption when compared to the resting condition and hydrated protocols. In line with this, serum iFABP levels were the highest with the strenuous dehydrated protocol (1443.72 μmol/L ± 249.9, p < 0.001), followed by the high intensity exercise at 70% Wmax in the hydrated condition., Conclusions: Exercise induces an increase in plasma citrulline, irrespective of a glutamine bolus. The extent to which this occurs is dependent on exercise intensity and the hydration state of the subjects. The same holds true for both the post-exercise increase in citrulline levels following glutamine supplementation and serum iFABP levels. These data indicate that citrulline release during exercise and after an oral glutamine bolus might be dependent on the intestinal health state and therefore on intestinal functionality. Glutamine is known to play a major role in intestinal physiology and the maintenance of gut health and barrier function. Together, this suggests that in clinical practice, a glutamine bolus to increase citrulline levels after exercise might be preferable compared to supplementing citrulline itself. To our knowledge this is the first time that exercise workload-related effects on plasma citrulline are reported in relation to intestinal damage., (Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x). Four laboratories received 13 coded sensitizers. Reproducible results were obtained in each laboratory. A binary prediction model, EC50≥7 mg/ml=weak to moderate sensitizer and EC50<7 mg/ml=strong to extreme sensitizer had an accuracy of 77%. A superior EE (EC50 and IL-1α2x) correlation was observed with human in vivo DSA05 data compared to LLNA-EC3 data. Human in vivo NOEL and LLNA-EC3 data correlated to a similar extent to in vitro EE data. Our results indicate that this easily transferable EE potency assay is suitable for testing chemical allergens of unknown potencies and may now be ready for further validation, providing complementary potency information to other assays already undergoing validation for assessing skin sensitization potential.

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The dopamine transporter (DAT) is responsible for the re-uptake of dopamine into presynaptic nerve terminals and thereby controls dopaminergic neurotransmission. Deletion of DAT results in a hyperdopaminergic phenotype and DAT(-/-) mice are characterized by pituitary hypoplasia, impaired maternal behavior, and increased locomotion. From earlier studies, we have evidence that the activity of the central dopaminergic system may play a role in determining immune reactivity and disease susceptibility. To further explore the functional relation between the dopaminergic system and the immune system, we investigated the activity of the immune system in DAT(-/-) mice. We show that in vitro, splenocytes from DAT(-/-) mice displayed reduced natural killer cell activity and reduced mitogen-induced cytokine responses. In contrast, LPS-induced cytokine production by macrophages was enhanced. In vivo, the cellular response to immunization with ovalbumine (OVA-induced delayed type hypersensitivity response) was significantly reduced. Interestingly, the OVA-induced humoral response (anti-OVA IgG) was increased in DAT(-/-) mice compared to wild-type animals. Plasma levels of catecholamines and corticosterone did not differ significantly between DAT(-/-) and wild-type animals. In conclusion, we show in the present study that interfering with the dopaminergic system has major consequences for both the acquired and the innate immune response.

There are large individual differences in cancer progression and it has been suggested that behavioral and psychological characteristics of cancer patients may contribute to the course of the disease. To get more insight in the contribution of behavioral characteristics to cancer progression, we investigated in rats, whether a stable behavioral trait characteristic is associated with NK cell activity, tumor angiogenesis, and tumor metastasis formation. Lewis rats were characterized based on locomotor activity in an open field. Rats in the upper and lower quartile were designated as high and low active rats. Low active animals had higher NK cell activity compared to their high active counterparts. In addition, we examined tumor angiogenesis by using a subcutaneous Matrigel implant containing MADB106 adenocarcinoma cells. Tumor Matrigel implants from low active animals contained significantly more hemoglobin compared to implants from high active animals, indicating a more pronounced angiogenic response in the low active animals. Finally, experimental lung metastasis formation was investigated by injecting MADB106 cells into the tail vein. Low active animals tended to develop more metastases. Moreover, low active animals developed significantly more tumors with a diameter larger than 2 mm, which is in line with higher angiogenic capacity. In conclusion, we demonstrated that individual differences in a stable behavioral trait are linked to individual differences in angiogenic capacity and tumor progression.

Interactions between the nervous system and the immune system have been recognized as important regulatory processes in determining the activity of the immune response. We have previously shown that rats, which differ in the reactivity of the dopaminergic system (APO-SUS and APO-UNSUS rats), also differ in experimental metastasis formation and in susceptibility to autoimmunity. APO-SUS rats have a high response to administration of apomorphine and can be characterized as hyperdopaminergic, whereas their APO-UNSUS counterparts show low susceptibility to apomorphine and have a hypodopaminergic phenotype. In this study we investigated whether the decreased experimental metastasis formation of APO-SUS rats compared to APO-UNSUS rats is associated with higher natural killer cell activity in APO-SUS rats. Surprisingly, splenic NK cell activity of hyperdopaminergic APO-SUS female as well as male rats is significantly lower than NK cell activity of their hypodopaminergic APO-UNSUS counterparts. The reduced splenic NK activity of female APO-SUS rats is associated with lower percentages of NK cells in the spleen cell population. In contrast, male APO-SUS and APO-UNSUS rats show similar numbers of NK cells in the spleen. There was no difference in plasma dopamine levels between APO-SUS and APO-UNSUS rats and i.p. treatment of rats with the dopaminergic agonist quinpirole did not alter NK cell activity. In conclusion, our data demonstrate that differences in the reactivity of the dopaminergic system are associated with differences in splenic NK cell activity. Moreover, our data demonstrate that in this model lower splenic NK cell activity is not related to increased experimental lung metastasis formation.

Maternal deprivation of neonatal animals has been shown to induce long-lasting changes in the reactivity of the neuroendocrine system. The aim of the present study was to investigate whether maternal deprivation also affects susceptibility to immune-mediated diseases such as experimental autoimmune encephalomyelitis (EAE) in adult life. To this end, 9-day-old rat pups were subjected to a short-lasting maternal deprivation for a period of 24 h. At the age of 8 weeks, we induced EAE in these rats by immunization with myelin basic protein (MBP) in complete Freund's adjuvant. Our data demonstrate that short-lasting maternal deprivation induces a marked increase in the severity of EAE in the animals in later life. The histopathological evaluation of spinal cord and cerebellum corresponded with the observed differences in clinical symptoms of EAE. Moreover, neonatal maternal deprivation affects macrophage functioning at adult age. In contrast, no differences were observed in in vitro mitogen- and MBP-induced cytokine production by splenocytes. LPS-induced corticosterone release did not differ either between maternally deprived and control animals. We conclude that short-lasting neonatal maternal deprivation of rat pups has long-lasting consequences for macrophage activity and for susceptibility to the inflammatory autoimmune disease EAE.

Outgrowth of solid tumors requires blood supply to the tumor. Tumor angiogenesis is dependent on the interplay between tumor-derived angiogenic factors and stromal cells. Recently, it has been shown that the neurotransmitter dopamine is a potent inhibitor of VEGF-induced angiogenesis. Moreover, there is evidence that patients with schizophrenia have a hyperreactive dopaminergic system and are relatively protected from cancer. We hypothesized that hyperreactivity of the dopaminergic system is related to reduced angiogenesis and tumor development. Therefore, we investigated tumor growth and angiogenesis in two lines of Wistar rats with high (APO-SUS) or low (APO-UNSUS) dopaminergic reactivity. Subcutaneous implants of mammary adenocarcinoma cells (MADB106) in matrigel remained 35% smaller in APO-SUS rats than in APO-UNSUS rats (P<0.01). Moreover, APO-SUS rats developed less lung metastases after i.v. administration of MADB106 tumor cells. Furthermore, hemoglobin content (APO-SUS: 40.6+/-7.6; APO-UNSUS: 76.9+/-13 mg/dl, P<0.05) and expression of the endothelial determinant PECAM-1 in tumors from APO-SUS rats were reduced (APO-SUS: 37+/-18; APO-UNSUS 69+/-25 units, P<0.01), indicating that reduced angiogenesis is responsible for reduced tumor development in APO-SUS rats. These results suggest a novel link between dopaminergic reactivity, angiogenesis, and tumor development and may explain part of the individual differences in cancer progression.