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Bladder cancer (BCa) and prostate cancer (PCa) are genitourinary cancers which constitute significant health problems in men and in which environmental factors play an important role. Understanding the genetic susceptibility to BCa or PCa and occupational exposure is paramount to improving cancer prevention and early detection. The aim of this review article was to address the scientific evidence on the genetic risk factors and occupational exposure associated with the occurrence of BCa and PCa. The authors identified relevant original articles that have been published between 1994 and 2023. Variations of the following search terms: "gene" and "occupational" combined with one of the following terms: "bladder cancer" or "prostate cancer" were applied for the search purpose. The authors found 342 publications of which 50 population studies met their requirements for gene-occupation interactions. In total, 34 full-text manuscripts were about BCa and 16 about PCa. These research examines the genes involved in detoxification processes of xenobiotics (glutathione S-transferase, N-acetyltransferase, cytochrome P450, UDP-glucuronosyltransferase), oxidative stress (glutathione peroxidase 1, manganese superoxide dismutase, catalase), altering DNA repair capacity (X-ray repair cross-complementing 1, base excision repair, nucleotide excision repair), tumour suppression (TP53 gene), and vitamin D pathway (vitamin D receptor gene). The role of genetic factors in the occupational exposure has not been conclusively established, but it appears the possibility of genetic involvement. Determination of environmentally responsive genes provides important mechanistic implications for the etiology of occupational cancers, and valuable input in occupational exposure limits set by taking genetic susceptibility into account. More genetic research is needed to corroborate these findings and assess their significance in the workplace. Med Pr. 2023;74(2):127-44.

Validated in vitro assays for testing non-genotoxic carcinogenic potential of chemicals are currently not available. Consequently, the two-year rodent bioassay remains the gold standard method for the identification of these chemicals. Transcriptomic and proteomic analyses have provided a comprehensive understanding of the non-genotoxic carcinogenic processes, however, functional changes induced by effects at transcriptional and translational levels have not been addressed. The present study was set up to test a number of proposed in vitro biomarkers of non-genotoxic hepatocarcinogenicity at the functional level using a translational 3-dimensional model. Spheroid cultures of human hepatocytes and stellate cells were exposed to 5 genotoxic carcinogenic, 5 non-genotoxic carcinogenic, and 5 non-carcinogenic chemical compounds and assessed for oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, apoptosis, and inflammation. The spheroid model could capture many of these events triggered by the genotoxic carcinogenic chemicals, particularly aflatoxin B1 and hydroquinone. Nonetheless, no clear distinction could be made between genotoxic and non-genotoxic hepatocarcinogenicity. Therefore, spheroid cultures of human liver cells may be appropriate in vitro tools for mechanistic investigation of chemical-induced hepatocarcinogenicity, however, these mechanisms and their read-outs do not seem to be eligible biomarkers for detecting non-genotoxic carcinogenic chemicals.

Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. This latter implies diagnostic difficulties, few therapeutic options and unclear mechanisms of action. To elucidate the metabolic alterations and pinpoint affected biochemical pathways, alcoholic steatohepatitis was simulated in vitro by exposing HepaRG cells to ethanol (IC10, 368 mM) and tumor necrosis factor alpha (TNF-α, 50 ng/mL) for 24 h. This combined exposure was compared to solely ethanol-exposed as well as -nonexposed cells. Four different metabolomics platforms were used combining liquid chromatography, high-resolution mass spectrometry and drift tube ion mobility to elucidate both intracellular and extracellular metabolic alterations. Some of the key findings include the influence of TNF-α in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylethanolamines and phosphatidylcholines. S-Adenosylmethionine showed to play a central role in the progression of alcoholic steatohepatitis. In addition, fatty acyl esters of hydroxy fatty acid (FAHFA)-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethoxylated phosphorylcholine was identified as a potential new biomarker of ethanol exposure.

BACKGROUND: It is unclear whether the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 C-to-G single nucleotide polymorphism, resulting in the substitution of isoleucine to methionine at position 148 (I148M), impedes regression of hepatic steatosis when treating non-alcoholic fatty liver disease (NAFLD). OBJECTIVES: Investigate if carriage of the PNPLA3 148M allele affects the anti-steatotic efficacy of all possible anti-NAFLD interventions, identify gaps in current knowledge and provide guidance for individual treatment. METHODS: Research available in public databases was searched up to 13 November 2022. Studies were included if a treatment in NAFLD patients decreased hepatic steatosis in the pooled patient group or a PNPLA3 I148M polymorphism subgroup (II/IM/MM). The risk of bias was assessed using the Cochrane Risk-Of-Bias 2 Tool and the Newcastle-Ottawa Scale. RESULTS: Moderate evidence indicates that NAFLD patients homozygous for the PNPLA3 148M allele benefit less or not at all from omega-3 carboxylic acids to decrease liver fat, while the PNPLA3 148I allele shows moderate benefit. Low evidence suggests that interventions employing lifestyle changes are more effective to reduce liver fat in NAFLD patients homozygous for the PNPLA3 148M allele compared to patients with wild-type PNPLA3. CONCLUSIONS: NAFLD patients homozygous for the PNPLA3 148M allele might not benefit from omega-3 carboxylic acids to reduce hepatic steatosis in contrast to patients with wild-type PNPLA3. Instead, patients with two PNPLA3 148M alleles should be especially advised to adopt lifestyle changes. Genotyping for PNPLA3 I148M should be encouraged in therapeutic studies for NAFLD. REGISTRATION NUMBER (PROSPERO): CRD42022375028.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA’s 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU ‘on the ground’ in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Peptide-based hydrogels represent promising systems for the sustained release of different types of drugs, ranging from small molecules to biologicals. Aiming at subcutaneous injection, which is a desirable parenteral administration route, especially for biologicals, we herein focus on physically crosslinked systems possessing thixotropic behaviour. The purpose of this study was to evaluate the in vitro and in vivo properties of hydrogels based on the amphipathic hexapeptide H-FQFQFK-NH2, which served as the lead sequence. Upon doubling the length of this peptide, the dodecapeptide H-FQFQFKFQFQFK-NH2 gave a significant improvement in terms of hydrogel’s in vivo stability post-injection, as monitored by nuclear SPECT/CT imaging. This increased hydrogel stability also led to a more prolonged in vivo release of encapsulated peptide cargoes. Even though no direct link with the mechanical properties of the hydrogels before injection can be made, an important effect of the subcutaneous medium was noticed in post invivo injection measurements. The results were validated for a therapeutically relevant analgesic peptide using the hot-plate test as an acute pain model. Gratifyingly, it was confirmed that elongation of the hydrogelator sequence induced more extended antinociceptive effects. Altogether, this simple structural modification of the hydrogelating peptide could provide a basis for reaching longer durations of action upon use of these soft biomaterials.

Previous studies have shown that differences in experimental design factors may alter the potency of genotoxic compounds in in vitro genotoxicity tests. Most of these studies used traditional statistical methods based on the lowest observed genotoxic effect levels, whereas more appropriate methods, such as the benchmark dose (BMD) approach, are now available to compare genotoxic potencies under different test conditions. We therefore investigated the influence of two parameters, i.e. cell type and exposure duration, on the potencies of two known genotoxicants [aflatoxin B1 and ethyl methanesulfonate (EMS)] in the in vitro micronucleus (MN) assay and comet assay (CA). Both compounds were tested in the two assays using two cell types (i.e. CHO-K1 and TK6 cells). To evaluate the effect of exposure duration, the genotoxicity of EMS was assessed after 3 and 24 h of exposure. Results were analyzed using the BMD covariate approach, also referred to as BMD potency ranking, and the outcome was compared with that of more traditional statistical methods based on lowest observed genotoxic effect levels. When comparing the in vitro MN results obtained in both cell lines with the BMD covariate approach, a difference in potency was detected only when EMS exposures were conducted for 24 h, with TK6 cells being more sensitive. No difference was observed in the potency of both EMS and aflatoxin B1 in the in vitro CA using both cell lines. In contrast, EMS was more potent after 24 h exposure compared with a 3 h exposure under all tested conditions, i.e. in the in vitro MN assay and CA in both cell lines. Importantly, for several of the investigated factors, the BMD covariate method could not be used to confirm the differences in potencies detected with the traditional statistical methods, thus highlighting the need to evaluate the impact of experimental design factors with adequate approaches.

Connexin proteins oligomerize in hexameric structures called connexin hemichannels, which then dock to form gap junctions. Gap junctions direct cell-cell communication by allowing the exchange of small molecules and ions between neighboring cells. In this way, hepatic gap junctions support liver homeostasis. Besides serving as building blocks for gap junctions, connexin hemichannels provide a pathway between the intracellular and the extracellular environment. The activation of connexin hemichannels is associated with acute and chronic liver pathologies. This article discusses the role of gap junctions and connexin hemichannels in the liver. © 2022 American Physiological Society. Compr Physiol 12:1-17, 2022.

The current study shows the research on phytochemical concerning the phenolic components of Algerian pomegranate fruit extract (Punica granatum L.) using Ultra Performance Liquid Chromatography Diode-Array Detection Electrospray Ionization Mass Spectrometry (UPLC-DAD-ESI-MS). In this study; about more than 50 phenolic compounds, including 40 phenolic acids, 5 flavonoids, 2 coumarins and ellagitannins were identified. The profile is characterized by the presence of major peak at retention time 0.59, related to Bergaptan diglucoside (40%). This latter compound and many compounds such as digalloyl lactone citric acid, dicafeoyl galloyl dihydroxymalonic acid, ferruloyl diglucosyl citrate, dicafeoyl galloyl malonyl citrate, protocatechic cafeoyl coumaroyl rhamnoside, isorhamnetin glucosyl rhamnoside, dihydrokaemferol ferruloyl cafeoyl, and bergapten triglucoside methylsulphide were first time reported and identified in the hydroalcoholic fruit extract of Algerian Punica granatum. This acquired information may be useful to validate the fruit extract of Punica granatum, as source of bioactive compounds.

Lipidomics analysis of zebrafish tissues has shown promising results to understand disease-related outcomes of exposure to toxic substances at molecular level. However, knowledge about their lipidome is limited, as most untargeted studies only identify the lipids that are statistically significant in their setup. In this work, liquid chromatography-high resolution mass spectrometry was used to study different aspects of the analytical workflow, i.e., extraction solvents (methanol/chloroform/water (3/2/2, v/v/v), methanol/dichloromethane/water (2/3/2, v/v/v) and methanol/methyl-tert-butyl ether/water (3/10/2.5, v/v/v), instrumental response, and strategies used for lipid annotation. The number of high-quality features (relative standard deviation of the intensity values ≤ 10% in the range 103−107 counts) was affected by the dilution of lipid extracts, indicating that it is an important parameter for developing untargeted methods. The workflows used allowed the selection of a dilution factor to annotate 712 lipid species (507 bulk lipids) in zebrafish liver using four software (LipidMatch, LipidHunter, MS-DIAL and Lipostar). Retention time mapping was a valuable tool to filter lipid annotations obtained from automatic software annotations. The lipid profiling of zebrafish livers will help in a better understanding of the true constitution of their lipidome at the species level, as well as in the use of zebrafish in toxicological studies.

Firefighters are exposed via multi-route exposure to a multitude of chemicals (PAHs, VOCs, flame retardants, dioxins, etc.) that may cause acute and long-term health effects. The dermal absorption of contaminants is a major contributor to the overall exposure and can be reduced by wearing appropriate personal protective equipment. As leather firefighters’ gloves cannot be decontaminated regularly by wet cleaning, many Belgian firefighters wear supplementary undergloves made of nitrile butadiene rubber (NBR) to protect against the accumulation of toxicants. However, the safety of this practice has been questioned. In this commentary, the current practice and risks are outlined for the first time, assessed by an interdisciplinary working group of the Belgian Superior Health Council. As NBR sticks to the skin more at high temperatures, the contact time on removal will be prolonged, posing an additional risk for deeper burns. However, based on the physicochemical properties of NBR and the existing experience of firefighters and burn centers, it is estimated that such incidents occur relatively rarely in practice. On the other hand, the risk of repeated exposure to contaminated gloves if no undergloves are worn is unacceptable. Despite the slightly increased risk for deeper burns, it is concluded that wearing disposable NBR gloves under regular firefighters’ gloves is an appropriate and effective preventive measure against toxic contamination. The nitrile butadiene rubber must always be fully covered to avoid any contact with the heat. ispartof: Toxics vol:11 issue:6 ispartof: location:Switzerland status: published

Due to a combination of rapid disease progression and the lack of curative treatment options, hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Infiltrated, monocyte-derived, tumor-associated macrophages are known to play a role in HCC pathogenesis, but the involvement of Kupffer cells (KCs) remains elusive. Here, we used the Clec4F-diphteria toxin receptor transgenic mouse model to specifically investigate the effect of KC depletion on HCC initiation, progression and neoplastic growth following liver resection. For this purpose, several HCC mouse models with varying underlying etiologies were used and partial hepatectomy was performed. Our results show that in HCC, developed on a fibrotic or non-alcoholic steatohepatitis background, depletion of embryonic KCs at the onset of HCC induction and the subsequent replacement by monocyte-derived KCs does not affect the tumor burden, tumor microenvironment or the phenotype of isolated KCs at end-stage disease. In non-chronic liver disease-associated diethylnitrosamine-induced HCC, ablation of Clec4F+ KCs did not alter tumor progression or neoplastic growth following liver resection. Our results show that temporal ablation of resident KCs does not impact HCC pathogenesis, neither in the induction phase nor in advanced disease, and indicate that bone marrow-derived KCs are able to swiftly repopulate the available KC niche and adopt their phenotype.

Background and aims: In a clinical intensive care setting, parenteral nutrition (PN) is often associated with liver dysfunction although other contributing factors such as sepsis, acute heart failure (AHF) and hepatotoxic drugs can be present. This retrospective study aimed to identify the impact of these factors on liver test dysfunction with focus on PN. Methods: Adult ICU patients admitted for ≥3 days and treated with PN for ≥ 3days were included. Liver test disturbances were recorded at day 1 of PN based on elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) or alkalic phosphatase (AP). The presence of AHF, sepsis and 5 previously selected hepatotoxic drugs was recorded. Volume of PN, calorie abundance, amount of proteins, lipids and carbohydrates were monitored. A linear mixed-effect model was used to assess the contribution of PN, sepsis, AHF and drugs to liver test abnormalities. Nutritional adequacy was defined as the caloric, protein, lipid and carbohydrate intake compared to theoretical needs. Results: Liver test abnormalities, presence of sepsis and AHF were the main confounders in predicting liver parameters in 224 ICU patients treated with at least 3 days PN. Presence of sepsis caused an average increase of 43% ± 7% for total bilirubin (TB), 36% ± 14% for gamma-glutamyltransferase (GGT) and 32% ± 8% for AP. Secondly, presence of AHF caused an increase of 29 ± 13% % for ALT and 75% ± 14% for AST. Volume of PN administrated caused an increase of 10% ± 5% for ALT and 14% ± 1% for AST. Carbohydrate intake exceeded nutritional recommendations and protein and lipid intake was not sufficient. Conclusion: Liver test disturbances in critically ill patients on PN for at least 3 days have multifactorial associations with sepsis and AHF being the main confounders. Volume of PN and hepatotoxic drugs had a smaller impact. Feeding adequacy revealed opportunity for improvement by reducing carbohydrate and increasing lipid and protein content.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available–and are currently in the process of regulatory validation–there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Primary sclerosing cholangitis (PSC) is an idiopathic chronic immune-mediated cholestatic liver disease characterized by fibro-inflammatory bile duct strictures, progressive hepatobiliary fibrosis, and gut-liver axis disruption. The pathophysiology of PSC remains insufficiently characterized, which hampers the development of effective therapies. Hepatic macrophages (MFs) such as Kupffer cells (KCs) are implicated in PSC pathogenesis, but their exact role is unclear. Using the latest markers to discriminate resident KCs (ResKCs) from their monocyte-derived counterparts (MoKCs), and two models of intrahepatic and extrahepatic cholestasis, respectively, this study showed that CLEC4F+TIM4+ ResKCs were depleted after chronic cholestatic liver injury. The infiltrating CLEC4F+TIM4- MoKCs were already enriched during the acute phase of PSC. Transcriptional profiling of hepatic MF subsets during early cholestatic injury indicated that ResKCs were indeed activated and that MoKCs expressed higher levels of pro-inflammatory and proliferative markers compared with those of ResKCs. As indicated in experiments with Clec4fDTR transgenic mice, conditional depletion of KCs, before and during early cholestasis induction, had no effect on the composition of the hepatic myeloid cell pool following injury progression and did not affect disease outcomes. Taken together, these results provide new insights into the heterogeneity of the MF pool during experimental PSC and evidence that depletion of resident and activated KCs during sclerosing cholangitis does not affect disease outcome in mice.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal–cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Despite extensive research and multiple clinical trials, there are still no FDA-approved therapies to treat the most severe forms of NAFLD. This is largely due to its complicated etiology and pathogenesis, which involves visceral obesity, insulin resistance, gut dysbiosis, etc. Although inflammation is generally believed to be one of the critical factors that drive the progression of simple steatosis to nonalcoholic steatohepatitis (NASH), the exact type of inflammation and how it contributes to NASH pathogenesis remain largely unknown. Liver inflammation is accompanied by the elevation of inflammatory mediators, including cytokines and chemokines and consequently intrahepatic infiltration of multiple types of immune cells. Recent studies revealed that extracellular vesicles (EVs) derived from inflammatory cells and hepatocytes play an important role in controlling liver inflammation during NASH. In this review, we highlight the roles of innate and adaptive immune cells and their microRNA-enriched EVs during NAFLD development and discuss potential drugs that target inflammatory pathways for the treatment of NAFLD.

Many guidelines offer recommendations to support the continuation of breastfeeding and the choice of medication when a mother undergoes a surgical procedure. The aim of this study is to investigate health care providers’ (HCPs) current practices and knowledge about peri-surgical medication and practices in breastfeeding women. We performed a cross-sectional study in Flanders (Belgium) assessing demographics, beliefs about breastfeeding and its health benefits, current practices concerning breastfeeding women undergoing (surgical) procedures and specific knowledge about the use of medication during breastfeeding. Two hundred and ninety-one (291) participants completed the online questionnaire. Many participants considered their knowledge about breastfeeding to be good, and almost all participants acknowledged the superiority of breastfeeding and the importance of its continuation. Very few participants were, however, familiar with the available protocols concerning surgical procedures in breastfeeding women. Less than half of the participants routinely advised the recommended practices to protect breastfeeding. For most of the peri-surgical medication, participants needed to look-up information about the compatibility with breastfeeding. We conclude that there is a knowledge gap and recommend the development of a comprehensive guideline as well as implementation of this information in basic and post-academic training.

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), scientific opinion on Butylated hydroxytoluene (BHT), preliminary version of September 27, 2021, final version of December 2, 2021, SCCS/1636/21.

Nonalcoholic fatty liver disease (NAFLD) encompasses nonalcoholic steatohepatitis (NASH) and affects 25% of the global population. Although a plethora of experimental models for studying NASH have been proposed, still scarce findings regarding the hepatic metabolomic/molecular profile. In the present study, we sought to unravel the hepatic metabolomic profile of mice subjected to a hybrid model of NASH, by combining a Western diet and carbon tetrachloride administration, for 8 weeks, in male C57BL/6J and BALB/c mice. In both mouse strains, the main traits of NASH—metabolic (glucose intolerance profile), morphologic (extensive microvesicular steatosis and fibrosis, lobular inflammation, and adipose tissue-related inflammation/hypertrophy), and molecular (impaired Nrf2/NF-κB pathway dynamics and altered metabolomic profile)—were observed. The hepatic metabolomic profile revealed that the hybrid protocol impaired, in both strains, the abundance of branched chain-aromatic amino acids, carboxylic acids, and glycosyl compounds, that might be linked to the Nrf2 pathway activation. Moreover, we observed a strain-dependent hepatic metabolomic signature, in which the tricarboxylic acid metabolites and pyruvate metabolism were dissimilarly modulated in C57BL/6J and BALB/c mice. Thus, we provide evidence that the strain-dependent hepatic metabolomic profile might be linked to the distinct underlying mechanisms of NASH, also prospecting potential mechanistic insights into the corresponding disease.

The neutral red uptake (NRU) assay is a cell viability assay that can be used for the assessment of compound-induced cytotoxicity. It is based on the ability of living cells to incorporate neutral red, a weak cationic dye, in lysosomes. The quantification of xenobiotic-induced cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red when compared to cells exposed to corresponding vehicle controls. The NRU assay is mainly used for hazard assessment in in vitro toxicology applications. Hence, this method has been incorporated in regulatory recommendations such as the OECD test guideline TG 432, in which an in vitro 3T3-NRU-phototoxicityassay is described to assess the cytotoxicity of compounds in the presence or absence of UV light.This book chapter describes a detailed protocol to carry out the NRU assay using the human hepatoma cell line HepG2, which is frequently employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetylsalicylic acid is assessed.

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Biolistic intracellular delivery of functional macromolecules makes use of dense microparticles which are ballistically fired onto cells with a pressurized gun. While it has been used to transfect plant cells, its application to mammalian cells has met with limited success mainly due to high toxicity. Here we present a more refined nanotechnological approach to biolistic delivery with light-triggered self-assembled nanobombs (NBs) that consist of a photothermal core particle surrounded by smaller nanoprojectiles. Upon irradiation with pulsed laser light, fast heating of the core particle results in vapor bubble formation, which propels the nanoprojectiles through the cell membrane of nearby cells. We show successful transfection of both adherent and non-adherent cells with mRNA and pDNA, outperforming electroporation as the most used physical transfection technology by a factor of 5.5–7.6 in transfection yield. With a throughput of 104-105 cells per second, biolistic delivery with NBs offers scalable and highly efficient transfections of mammalian cells.

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endome-trial microbiome is of particular interest given the potential impact on the embryo implanta-tion. To date, all endometrial microbiome studies have applied a metagenomics approach. A se-quencing-based technique, however, has its limitations, more specifically in adequately explor-ing low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hys-teroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were includ-ed after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial mi-crobiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab®-assisted culturomics to inves-tigate the low biomass endometrial microbiome at a species level.

Ipomoea aquatica is a common green leafy vegetable that has numerous uses in traditional medicine. This study focused on the determination of the cytotoxic, antiradical, and antidiabetic properties of various fractions of the I. aquatica methanolic extract, as well as on the tentative identification of some bioactive compounds in the same fractions. The cytotoxicity was determined by the brine shrimp lethal test. The antioxidant activities of the I. aquatica fractions were investigated through 3 assays. The antidiabetic activity (in vitro) was measured by α-glucosidase and α-amylase inhibition assays. Phytochemical qualitative analyses demonstrated the presence of alkaloids, terpenoids, phenols, and flavonoids in the ethyl acetate-methanol and methanol fractions. The total phenolic and total flavonoid contents were found to be highest in the ethyl acetate-MeOH fractions. The evaluation of the cytotoxicity showed that the hexane-dichloromethane fraction is the most toxic, while the others are moderately toxic. The antioxidant activity assays showed that the ethyl acetate-MeOH fractions are the most potent, while the α-glucosidase and α-amylase assays revealed that the hexane-dichloromethane fraction might contain a potent antidiabetic agent. Some bioactive substances in the MeOH fractions, such as salicylic acid glucoside, 1-O-sinapoyl-β-D-glucose derivative, and dihydroferulic acid derivative, were tentatively identified. To the best of our knowledge, this is the first report to detect and identify these compounds in this species. Based on the results of this study, it may be concluded that I. aquatica is a potent antioxidant agent and could be a good candidate as a natural antioxidant in food and therapeutics.

Background: It is well known, although very understudied, that the male reproductive system is the target of many substances like endocrine disruptors [1, 2]. However, only animal models are used in reprotoxicology [3], making it harder for translation of results to the human. Thus, an effective and more relevant in vitro model from human tissue is in great demand. Testicular organoids can play an important role in the search for such a model [4], but prepubertal testis tissue, the most efficient in organoid formation [5, 6], is rare and scarce. Hence, we developed a platform that uses an alternative, readily available testis tissue and a culture system that allows upscaled and easy production of human testicular organoids (an important feature in toxicity testing). Methods: Primary testis cells from five donors were cultured in a stem cell medium for two weeks in microwells. Several cell densities were tested for improved organoid formation. Immunofluorescence staining for specific markers was done to identify the location of the main testicular cell types and extracellular matrix proteins in the organoids. Additionally, a 7-day culture was started to collect a set of organoids each day and stain them for the main testicular cells mapping the reorganisation process of the organoids. Results: The main testicular cells were present in the organoids: Sertoli cells, spermatogonia, peritubular myoid cells, and Leydig cells. Testicular cells of most donors showed the ability to self-assemble into organoids, with clear compartmentalisation – core composed of peritubular myoid cells and extracellular matrix proteins, and lining periphery of Sertoli cells and Leydig cells. When these results were compared with the original tissue histology, it was observed that the tissues that resulted in clear organoid compartmentalisation were also those that seemed to have a higher Sertoli cell density. Moreover, seeding 28 000 to 43 000 cells/microwell resulted in better organisation and tissue viability. When mapping the organoid organisation, dissociated cells create first a mixed cluster and then, after approximately 3 days, the peritubular myoid cells have migrated to the centre of the organoid creating a tight core. Conclusions: Cells from the alternative, readily available testis tissue used can self-assemble into organoids like those from prepubertal testis tissue. Although the herein presented testicular organoids display a compartmentalised organisation, this organisation is not yet like the one in vivo (peritubular myoid cells around seminiferous epithelium). Notwithstanding, this is a model that has the potential to be used in testis toxicological assays. Future research should investigate the origin of interpersonal variation in organoid performance in vitro; differentiation of spermatogonia; and the organoids’ degree of physiomimicry.

Pannexin1 channels facilitate the extracellular release of a number of messengers, including adenosine triphosphate. Although fulfilling some physiological functions, pannexin1 channel communication has to date been primarily studied in the context of inflammation and cell death. In the past decade, a variety of chemical substances have been reported to induce pannexin1 channel opening, including metals and chelating agents, particulate matter, nanoparticles and drugs. While the pathophysiological aspects of pannexin1 channel communication have been reviewed on many previous occasions, the present paper intends to provide a short perspective in order to motivate research that will advance mechanistic understanding of the roles of pannexin1 signaling in chemical toxicity.

Background: Healthcare-associated SARS-CoV-2 infections need to be explored further. Our study is an analysis of hospital-acquired infections (HAIs) and ambulatory healthcare workers (aHCWs) with SARS-CoV-2 across the pandemic in a Belgian university hospital. Methods: We compared HAIs with community-associated infections (CAIs) to identify the factors associated with having an HAI. We then performed a genomic cluster analysis of HAIs and aHCWs. We used this alongside the European Centre for Disease Control (ECDC) case source classifications of an HAI. Results: Between March 2020 and March 2022, 269 patients had an HAI. A lower BMI, a worse frailty index, lower C-reactive protein (CRP), and a higher thrombocyte count as well as death and length of stay were significantly associated with having an HAI. Using those variables to predict HAIs versus CAIs, we obtained a positive predictive value (PPV) of 83.6% and a negative predictive value (NPV) of 82.2%; the area under the ROC was 0.89. Genomic cluster analyses and representations on epicurves and minimal spanning trees delivered further insights into HAI dynamics across different pandemic waves. The genomic data were also compared with the clinical ECDC definitions for HAIs; we found that 90.0% of the ‘definite’, 87.8% of the ‘probable’, and 70.3% of the ‘indeterminate’ HAIs belonged to one of the twenty-two COVID-19 genomic clusters we identified. Conclusions: We propose a novel prediction model for HAIs. In addition, we show that the management of nosocomial outbreaks will benefit from genome sequencing analyses.

The workshop titled "Application of evidence-based methods to construct mechanism-driven chemical assessment frameworks" was co-organized by the Evidence-based Toxicology Collaboration and the European Food Safety Authority (EFSA), and hosted by EFSA at its headquarters in Parma, Italy on October 2 and 3, 2019. The goal was to explore integration of systematic review methods with mechanistic evidence evaluation. Participants were invited to work on concrete products to advance the exploration of how evidence-based approaches can support the development and application of adverse outcome pathways (AOP) in chemical risk assessment. The workshop discussions were centered around three related themes: 1) assessing certainty in AOPs, 2) literature-based AOP development and 3) integrating certainty in AOPs and non-animal evidence into decision frameworks. Several challenges, mostly related to methodology, were identified, and largely determined the workshop recommendations. The workshop recommendations included the comparison and potential alignment of processes used to develop AOP and systematic review methodology, including the translation of vocabulary of evidence-based methods to AOP and vice versa, the development and improvement of evidence mapping and text mining methods and tools, as well as a call for a fundamental change in chemical risk and uncertainty assessment methodology if to be conducted based on AOPs and new approach methodologies (NAM). The usefulness of evidence-based approaches for mechanism-based chemical risk assessments was stressed, particularly the potential contribution of the rigor and transparency inherent to such approaches in building stakeholders' trust for implementation of NAM evidence and AOP into chemical risk assessment.

Knowledge bases store information on certain topics. Applying a well-structured and machine-readable format for a knowledge base is a prerequisite for any AI-based processing or reasoning. Semantic technologies (e.g. RDF) offer such a format and have the advantages that they make it possible to define the semantics of the information and support advanced querying. However, the disadvantage is that using such technologies is challenging for people not trained in IT, such as subject matter experts. This means that they need to rely on semantic technology experts to create, maintain, and query their knowledge bases. However, these experts are, in turn, not trained in the subject matter, while domain knowledge is essential for the construction of high-quality knowledge bases. In this paper, we present an end-user engineering approach for ontology-based knowledge bases. The goal is to allow subject matter experts to develop, maintain, and exploit the knowledge base themselves. We also present the supporting tools developed so far. The tools for the construction and the manual filling of the knowledge base are using the jigsaw metaphor to hide technicalities and guide the users. We also developed tools to automatically import data from spreadsheets into the knowledge base and to perform some type of quality control on the data. The end-user approach and the tools are demonstrated and evaluated for building a knowledge base in the toxicology domain.

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on Acid Yellow 3 - C054 (CAS Number 8004-92-0, EC No 305-897-5), submission II, preliminary version of 7 May 2021, final version of 23 July 2021, SCCS/1631/21.

Objectives Alcohol and medication use are increasingly prevalent in the older population. Concurrent use of alcohol and alcohol-interactive (AI) medication can lead to significant adverse consequences. Methods Three reference works were used to create an explicit list of drug substances for which information about the interaction with alcohol was available in at least one of them. Additional information was extracted from the Summary of Product Characteristics (SPC). The first aim was to generate a list of 256 substances with standardized advice regarding the concurrent use of each drug with alcohol. The second aim was to observe the prevalence of potential drug–alcohol-interactions. The list was applied to a database containing information about alcohol and medication use of 1,016 community-dwelling older patients (≥70 years) with polypharmacy. Results About half of the sample population reported to consume alcohol at least once a week. Around 22% were classified as frequent drinkers (5–7 days/week) and 11% as heavier drinkers (>7 units/week). Ninety-three percent alcohol consumers in our sample took at least one chronic drug that potentially interacts with alcohol and 42% used at least one chronic drug for which alcohol use is considered contraindicated. Conclusions We developed an explicit list of potentially drug–alcohol-interactions in older adults, with standardized handling advice. We observed that prevalence of potential drug–alcohol-interactions is substantial in community-dwelling older patients with polypharmacy.

Risk assessments of various types of chemical compounds are carried out in the European Union (EU) foremost to comply with legislation and to support regulatory decision-making with respect to their safety. Historically, risk assessment has relied heavily on animal experiments. However, the EU is committed to reduce animal experimentation and has implemented several legislative changes, which have triggered a paradigm shift towards human-relevant animal-free testing in the field of toxicology, in particular for risk assessment. For some specific endpoints, such as skin corrosion and irritation, validated alternatives are available whilst for other endpoints, including repeated dose systemic toxicity, the use of animal data is still central to meet the information requirements stipulated in the different legislations. The present review aims to provide an overview of established and more recently introduced methods for hazard assessment and risk characterisation for human health, in particular in the context of the EU Cosmetics Regulation (EC No 1223/2009) as well as the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation (EC 1907/2006).

Adverse outcome pathways (AOPs), introduced in modern toxicology, intend to provide an evidence-based representation of toxicological effects and facilitate safety assessment of chemicals not solely based on laboratory animal in vivo experiments. However, some toxicological processes are too complicated to represent in one AOP. Therefore, AOP networks are developed that help understanding and predicting toxicological processes where complex exposure scenarios interact and lead to the emergence of the adverse outcome. In this study, we present an AOP network for breast cancer, developed after an in-depth survey of relevant scientific literature. Several molecular initiating events (MIE) were identified and various key events that link the MIEs with breast cancer were described. The AOP was developed according to Organization of Economic Co-Operation and Development (OECD) guidance, weight of evidence was assessed through the Bradford Hill criteria and confidence was tested by the OECD key questions. The AOP network provides a straightforward understanding of the disease onset and progression at different biological levels. It can be used to pinpoint knowledge gaps, identify novel therapeutic targets and act as a stepping stone for the development of novel in vitro test methods for hazard identification and risk assessment of newly developed chemicals and drugs.

Background: Earlier, we showed the possibility of potency classification of promising peroxisome proliferator-activated receptor (PPAR)-agonists for anti-non-alcoholic steatohepatitis (NASH) treatment using human-derived hepatic in vitro disease models including primary human hepatocytes (PHH), human skin stem cell-derived hepatic progenitors (hSKP-HPC) and cancer cell lines (HepaRG, HepG2). Among those PPAR-agonists, elafibranor, followed by saroglitazar and pioglitazone showed the strongest anti-NASH potencies in vitro. Elafibranor, however, recently failed to show sufficient efficacy in a phase III clinical trial interim analysis and its development was discontinued. Therefore, we aimed to investigate possible molecular mechanisms that could lay at the basis of this failure. Methods: PHH, hSKP-HPC and HepaRG cultures were exposed to NASH-inducing triggers (insulin, glucose, sodium oleate, palmitic acid, TNF-α, IL-1β and TGF-β) with or without elafibranor for 24h. Hereafter, whole-genome microarrays (Affymetrix) were performed and used for pathway analysis. Publicly-available transcriptomic data of clinical samples of NASH patients and patients with resolved NASH after bariatric surgery served as benchmarks. Results: Hepatic ‘NASH-induced’ cultures showed up to 35% overlap with transcriptional signatures present in samples of NASH patients, indicating the relevance of the in vitro models. In all in vitro models, elafibranor restricted NASH-specific cellular processes through the activation and inhibition of processes also seen in pre- and post- bariatric surgery samples of NASH patients. PPARGC1A, PPARA and SIRT1 were identified as common upstream regulators in the three in vitro models exposed to elafibranor. Elafibranor induced upregulation of PLIN2, PDK4 and ANGPTL4, which have been earlier linked to liver steatosis, under regulation of the common upstream regulators. These pro-steatogenic mediators were, however, not increased in liver samples of patients with resolved NASH. Conclusions: Modulations of ANGPTL4, PDK4 and PLIN2 deserve further attention in the preclinical investigation of PPAR-agonists for the treatment of NASH.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term ‘the Three Rs’, which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years — not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA’s 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as ‘on the ground’ facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases.

Objectives: Non-alcoholic fatty liver disease (NAFLD) has a growing epidemiologic and economic burden. It is associated with Western diet (WD) patterns, and its pathogenesis involves metabolic disorders (obesity, dyslipidemia, hyperglycemia, and diabetes) and gut dysbiosis, features that are usually neglected or not reproduced by most animal models. Thus, we established a 6-mo WD-induced NAFLD mouse model associated with metabolic disorder, investigating its main features at the gut microbiome-liver-adipose tissue axis, also evaluating the correlations of gut dysbiosis to the other disease outcomes. Methods: Male C57 BL6 mice received a high-fat (30% lard and 0.2% cholesterol, 57% calories) and sucrose-rich (20%) chow, and a high-sugar solution (23.1 and 18.9 g/L of D-fructose and D-glucose) for 6 mo. Results: The model featured high serum cholesterol levels, glucose intolerance, and hyperinsulinemia. WD intervention resulted in extensive macro/microvesicular liver steatosis and pericellular fibrosis-resembling human disease-accompanied by hepatic stellate cell activation and CD68+ macrophage infiltration, increased protein levels of proinflammatory p65-nuclear factor-kB, interleukin-6 and tumor necrosis factora, with decreased antioxidant regulator Nrf2. Mice showed clear obesity with adipocyte hypertrophy, and CD68+macrophage/mast cell infiltration in adipose tissue while a reduction in number of goblet cells was also observed in the small intestine. Moreover, the pyrosequencing of the 16 S ribosomal RNA of gut cecal content showed decreased bacterial diversity, enriched Firmicutes and Proteobacteria, decreased Bacteroidetes and Fusobacteria, and increased ratio of Firmicutes to Bacteroidetes. Bacteroidetes and Bacteroides had the highest number of significant correlations with liver-adipose tissue axis outcomes. In silico analysis of gut microbiome in NAFLD obese patients revealed a depletion in Bacteroides, which also correlated to disease outcomes. Conclusion: This mice model gathered suitable phenotypical alterations in gut-liver-adipose tissue axis that resembled NAFLD associated with metabolic disorders in humans and may be considered for preclinical investigation.

In the original version of the article the images for Figs. 3 and 4 were interchanged, this has now been corrected so that the images and captions for Figs. 3 and 4 match.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:9 ispartof: location:Switzerland status: published

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project “Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework” aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and posi­tioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowd­sourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, inte­grating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

Cholestatic liver disease denotes any situation associated with impaired bile flow concomitant with a noxious bile acid accumulation in the liver and/or systemic circulation. Cholestatic liver disease can be subdivided into different types according to its clinical phenotype, such as biliary atresia, drug‐induced cholestasis, gallstone liver disease, intrahepatic cholestasis of pregnancy, primary biliary cholangitis and primary sclerosing cholangitis. Considerable effort has been devoted to elucidating underlying mechanisms of cholestatic liver injuries and explore novel therapeutic and diagnostic strategies using animal models. Animal models employed according to their appropriate applicability domain herein play a crucial role. This review provides an overview of currently available in vivo animal models, fit‐for‐purpose in modelling different types of cholestatic liver diseases. Moreover, a practical guide and workflow is provided which can be used for translational research purposes, including all advantages and disadvantages of currently available in vivo animal models.

Background & Aims: Liver fibrosis is a common consequence of chronic liver injury and is characterized by the accumulation of extracellular matrix mainly generated from activated hepatic stellate cells (HSCs). At present, the mechanisms underlying liver fibrogenesis remain obscure and effective pharmacological therapies are lacking. Neutrophil-specific microRNA-223 (miR-223) plays an important role in controlling the development of various liver diseases; however, its role in HSC activation and liver fibrosis remains unclear. Methods: Liver fibrosis was induced by chronic carbon tetrachloride (CCl4) injection of miR-223 knockout (miR-223KO) mice and littermate wild-type controls. MiR-223 was overexpressed in cultured HSCs to determine its function and targets during HSC activation and proliferation. The expression of miR-223 and pri-miR-223 was examined in primary HSCs isolated from CCl4-treated mice and in cultured HSCs. The communication between HSCs and neutrophils was studied by performing in vitro co-culture experiments. Results: Genetic deletion of miR-223 exacerbated chronic CCl4-induced liver fibrosis. Administration of miR-223 inhibited liver fibrosis by inhibiting the transcriptional coactivator with PDZ-binding motif (TAZ)-Indian hedgehog (IHH)-GLI Family Zinc Finger 2 (GLI2) pathway via the crosstalk between hepatocytes and HSCs. Overexpression of miR-223 also directly attenuated Gli2 as well as platelet-derived growth factor receptor α/β (Pdgfra/b) expression in HSCs, thereby suppressing HSC activation and proliferation. The expression of pri-miR-223 and miR-223 was downregulated during HSC activation in vitro. Expression of pri-miR-223 was also decreased in activated HSCs in vivo in fibrotic livers but mature miR-223 expression was not reduced. Finally, in co-culture experiments, activated HSCs were able to take up miR-223-enriched extracellular vesicles from neutrophils, resulting in elevation of miR-223. Conclusion: MiR-223 restricts liver fibrosis by targeting multiple genes in hepatocytes and HSCs, providing potential therapeutic targets for the treatment of liver fibrosis.