Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet

\ua9 2024 The AuthorsIn the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI

Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes

BackgroundDysbindin, a cytoplasmic protein long known to function in the biogenesis of specialized lysosome-related organelles (LROs), has been reported to reduce surface expression of D2 dopamine receptors in neurons. Dysbindin is broadly expressed, and dopamine receptors are members of the large family of G protein-coupled receptors (GPCRs) that function in diverse cell types. Thus we asked if dysbindin regulates receptor number in non-neural cells, and further investigated the cellular basis of this regulation.Methodology/principal findingsWe used RNA interference to deplete endogenous dysbindin in HEK293 and HeLa cells, then used immunochemical and biochemical methods to assess expression and endocytic trafficking of epitope-tagged GPCRs. Dysbindin knockdown up-regulated surface expression of D2 receptors compared to D1 receptors, as reported previously in neurons. This regulation was not mediated by a change in D2 receptor endocytosis. Instead, dysbindin knockdown specifically reduced the subsequent trafficking of internalized D2 receptors to lysosomes. This distinct post-endocytic sorting function explained the minimal effect of dysbindin depletion on D1 receptors, which recycle efficiently and traverse the lysosomal pathway to only a small degree. Moreover, dysbindin regulated the delta opioid receptor, a more distantly related GPCR that is also sorted to lysosomes after endocytosis. Dysbindin was not required for lysosomal trafficking of all signaling receptors, however, as its depletion did not detectably affect down-regulation of the EGF receptor tyrosine kinase. Dysbindin co-immunoprecipitated with GASP-1 (or GPRASP-1), a cytoplasmic protein shown previously to modulate lysosomal trafficking of D2 dopamine and delta opioid receptors by direct interaction, and with HRS that is a core component of the conserved ESCRT machinery mediating lysosome biogenesis and sorting.Conclusions/significanceThese results identify a distinct, and potentially widespread function of dysbindin in promoting the sorting of specific GPCRs to lysosomes after endocytosis

Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells

Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells

A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule

Adaptations to Endosymbiosis in a Cnidarian-Dinoflagellate Association: Differential Gene Expression and Specific Gene Duplications

Trophic endosymbiosis between anthozoans and photosynthetic dinoflagellates forms the key foundation of reef ecosystems. Dysfunction and collapse of symbiosis lead to bleaching (symbiont expulsion), which is responsible for the severe worldwide decline of coral reefs. Molecular signals are central to the stability of this partnership and are therefore closely related to coral health. To decipher inter-partner signaling, we developed genomic resources (cDNA library and microarrays) from the symbiotic sea anemone Anemonia viridis. Here we describe differential expression between symbiotic (also called zooxanthellate anemones) or aposymbiotic (also called bleached) A. viridis specimens, using microarray hybridizations and qPCR experiments. We mapped, for the first time, transcript abundance separately in the epidermal cell layer and the gastrodermal cells that host photosynthetic symbionts. Transcriptomic profiles showed large inter-individual variability, indicating that aposymbiosis could be induced by different pathways. We defined a restricted subset of 39 common genes that are characteristic of the symbiotic or aposymbiotic states. We demonstrated that transcription of many genes belonging to this set is specifically enhanced in the symbiotic cells (gastroderm). A model is proposed where the aposymbiotic and therefore heterotrophic state triggers vesicular trafficking, whereas the symbiotic and therefore autotrophic state favors metabolic exchanges between host and symbiont. Several genetic pathways were investigated in more detail: i) a key vitamin K–dependant process involved in the dinoflagellate-cnidarian recognition; ii) two cnidarian tissue-specific carbonic anhydrases involved in the carbon transfer from the environment to the intracellular symbionts; iii) host collagen synthesis, mostly supported by the symbiotic tissue. Further, we identified specific gene duplications and showed that the cnidarian-specific isoform was also up-regulated both in the symbiotic state and in the gastroderm. Our results thus offer new insight into the inter-partner signaling required for the physiological mechanisms of the symbiosis that is crucial for coral health

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field

Full-Length L1CAM and Not Its Δ2Δ27 Splice Variant Promotes Metastasis through Induction of Gelatinase Expression

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression

EVpedia: a community web portal for extracellular vesicles research

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research.X1110478Ysciescopu

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.