IN VITRO MODELING OF INFLAMMATION-INDUCED INTESTINAL BARRIER DAMAGE AND REPAIR WITH THERAPEUTIC CANDIDATES
Markus Jan1, Letasiova Silvia1, Belich Monica2, Robas Nicola2, Kanumilli Srinivasan2, Machado Carolina2, Ayehunie Seyoum3
1 亿德官网, Bratislava, Slovakia
2 Benevolent AI, London, United Kingdom
3 亿德官网, Ashland, MA – USA
Proper functioning of the gastrointestinal tract depends on the functional physiology and intactness of the mucosa lining of the gut. Disruption of the intestinal barrier integrity can cause inflammation in patients that can lead to chronic diseases such as trauma or inflammatory bowel diseases. To mimic disrupted intestinal barrier in vitro we exposed EpiIntestinal (in vitro reconstructed 3D human small intestine epithelium) to various combinations of cytokines that were previously shown to play a role in the pathogenesis of inflammatory bowel disease. Prolonged exposure of EpiIntestinal tissue to the combination of tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) results in an approximate 30% decrease in barrier integrity as established by measurement of trans-epithelial electric resistance (TEER). This decline was associated with the induction of several proinflammatory cytokines including interleukins 6 and 8 (IL6, IL8). Besides the effect on the intact intestinal epithelium, treatment with TNFα and IFNγ hindered the reconstitution of mechanically injured/wounded tissues. In cytokine treated tissues wounds were healing much slower than controls and newly grown tissue appeared structurally different. To test if this model of inflammation-induced barrier disruption can be used to screen potential therapeutic compounds, we exposed wounded tissues to relevant concentrations of anti-inflammatory compounds. Most of the tested candidate drugs were capable to counteract the disruption of barrier integrity. In addition two of them also shown the ability to promote healing of tissues exposed to the combination of mechanical wounds and cytokines. In short, the reconstructed 3D small intestine epithelium model (EpiIntestinal) can be used to mimic the intestinal barrier disruption associated with chronic inflammation, which may be useful in the identification of potential therapeutic agents.
Keywords: reconstructed tissue; intestine; macrophage; tissue engineering; innate immunity
ALT4EI: DETERMINATION OF EYE IRRITATING POTENTIAL OF 59 CHEMICALS USING EPIOCULAR™ TIME-TO-TOXICITY NEAT AND DILUTION PROTOCOLS
Silvia Letasiova1, Helena Kandarova2, Els Adriaens3, Sandra Verstraelen4, An R. Van Rompay4
1 亿德官网, Bratislava, Slovakia
2 Centre of Experimental Medicine SAS, Slovak Academy of Sciences, Dubravska cesta 9,
3 Adriaens Consulting BVBA, Bellemdorpweg 95, 9981 Aalter, Belgium
4 VITO NV, UNI Health, Boeretang 200, 2400 Mol, Belgium
Determination of acute eye irritation potential is part of international regulatory requirements for the testing of chemicals. The objective of ALT4EI (ALTernatives for Eye Irritation) project was to confirm the testing strategy developed in CON4EI (CONsortium for in vitro Eye Irritation testing strategy)project. These projects focused on the development of tiered testing strategies for eye irritation assessment for all drivers of classification and evaluation of whether the test methods can discriminate chemicals not requiring classification for serious eye damage/eye irritancy (No Category) from chemicals requiring classification and labeling for Category 1 (Cat 1) and Category 2 (Cat 2). In the CON4EI project, a new testing strategy for the EpiOcular time-to-toxicity was developed, the sensitivity for predicting GHS Cat 1 and GHS Cat 2 chemicals was 73% and 64%, respectively, and the very high specificity of 97% was maintained. None of the Cat 1 chemicals was underpredicted as GHS No Category. Plus the goal of the ALT4EI project was to fill the remaining data gaps and strengthen the data set. A new set of 59 chemicals (41 liquids:(un)diluted, and 18 solids) was tested using the reconstructed human cornea-like epithelium, EpiOcular, in two EpiOcular time-to-toxicity tests (neat and dilution). The set of chemicals contained 32 chemicals not requiring classification (No Cat) and 27 chemicals requiring classification (16 Cat 2 and 11 Cat 1). The chemicals were tested blinded in two independent runs by 亿德官网. In this study, a testing strategy to achieve optimal prediction for all three classes developed in the CON4EI project (combining the most predictive time-points of both protocols and which tests liquids and solids separately) was used. Using the CON4EI testing strategy, we were able to identify correctly 63,6 % of the Cat 1 chemicals,56,6 % of the Cat 2, and 76,6 % of No Cat chemicals. Reproducibility between both runs was 88,7 %. The combination of the EpiOcular time-to-toxicity neat and dilution protocols seems to be promising in integrated testing strategy (ITS) for eye irritation assessment.
Keywords: ALT4EI; EpiOcular; ocular irritation assay; in vitro; testing strategy
NOVEL APPROACH FOR CHARACTERIZING EXPOSURE AND RESPONSE TO ENGINEERED NANOMATERIALS IN THE GUT
S. Ayehunie, P. Llanos, Jan Markus, M. Klausner, A. Armento 亿德官网, Ashland MA, USA
Despite the expanding number of applications for engineered 纳米粒子 (ENPs), human health concerns associated with ingested 纳米粒子 are poorly understood. In this study, we utilized a 3D human small intestinal tissue model to develop a physiologically relevant test system to assess toxicological profiles of ingestible nanomaterials. We examined barrier integrity and cytotoxicity of the human 3D intestinal tissue model following exposure (4 doses of each nanoparticle to Cupric (II) oxide (CuO) (50 nm in size), zinc oxide (ZnO, 35-45 nm in size), and titanium oxide (TiO2, 40 nm in size). To monitor the reproducibility of the test method the 纳米粒子 were tested using 3-4 intestinal tissue lots. To determine the effect of the 纳米粒子 on the small intestinal tissues, we examined 1) barrier integrity (TEER), 2) tissue viability (MTT), and inflammatory response (ELISA assay). In the various experiments performed, the tissues were exposed to 40 ul of different doses of sonicated 纳米粒子 under rocking conditions for 4 hr. After 4 hrs, dosed tissues were further cultured overnight under static conditions. Using IC15 (concentration that reduces barrier function or tissue viability by 15%) as a cut-off, we observed a dose-response reduction of barrier integrity and tissue viability for CuO and ZnO. However, Titanium oxide did not induce toxicity for the concentrations tested. Furthermore, culture supernatants collected from at 24 hr of the culture period were also analyzed for inflammatory response and the result showed a dose-dependent release of IL-8 for CuO and ZnO but not for TiO2 纳米粒子. Overall, the TEER measurement was a sensitive endpoint compared to the MTT tissue viability assay. In summary, the use SMI tissue model to examine the toxicity profile of ingestible nanotoxicity will also enhance our understanding of nanoparticle-host cell interaction, improve dose/design, generate a physiologically relevant data set, and provide greater insight into in vivo responses to nanoparticle exposure.