The increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant Aspergillus fumigatus, is a life-threatening challenge to the immunocompromised population.... Show moreThe increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant Aspergillus fumigatus, is a life-threatening challenge to the immunocompromised population. Autophagy-related processes such as LC3-associated phagocytosis have been shown to be activated in the host response against fungal infection, but their overall effect on host resistance remains uncertain. To analyze the relevance of these processes in vivo, we used a zebrafish animal model of invasive Aspergillosis. To confirm the validity of this model to test potential treatments for this disease, we confirmed that immunosuppressive treatments or neutropenia rendered zebrafish embryos more susceptible to A. fumigatus. We used GFP-Lc3 transgenic zebrafish to visualize the autophagy-related processes in innate immune phagocytes shortly after phagocytosis of A. fumigatus conidia, and found that both wild-type and melanin-deficient conidia elicited Lc3 recruitment. In macrophages, we observed GFP-Lc3 accumulation in puncta after phagocytosis, as well as short, rapid events of GFP-Lc3 decoration of single and multiple conidia-containing vesicles, while neutrophils covered single conidia-containing vesicles with bright and long-lasting GFP-Lc3 signal. Next, using genetic and pharmacological stimulation of three independent autophagy-inducing pathways, we showed that the antifungal autophagy response improves the host survival against A. fumigatus infection, but only in the presence of phagocytes. Therefore, we provide proof-of-concept that stimulating the (auto)phagolysosomal pathways is a promising approach to develop host-directed therapies against invasive Aspergillosis, and should be explored further either as adjunctive or stand-alone therapy for drug-resistant Aspergillus infections. Show less
In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph... Show moreIn Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson's disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1-/- fish) and without GlcSph (gba1-/-:asah1b-/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1-/-:asah1b-/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons. Show less
To advance the systems approach in pharmacology, experimental models and computational methods need to be integrated from early drug discovery onward. Here, we propose outside‐in model development,... Show moreTo advance the systems approach in pharmacology, experimental models and computational methods need to be integrated from early drug discovery onward. Here, we propose outside‐in model development, a model identification technique to understand and predict the dynamics of a system without requiring prior biological and/or pharmacological knowledge. The advanced data required could be obtained by whole vertebrate, high‐throughput, low‐resource dose‐exposure‐effect experimentation with the zebrafish larva. Combinations of these innovative techniques could improve early drug discovery. Show less
The ability to culture complex organs is currently an important goal in biomedical research. It is possible to grow organoids (3D organ-like structures) in vitro; however, a major limitation of... Show moreThe ability to culture complex organs is currently an important goal in biomedical research. It is possible to grow organoids (3D organ-like structures) in vitro; however, a major limitation of organoids, and other 3D culture systems, is the lack of a vascular network. Protocols developed for establishing in vitro vascular networks typically use human or rodent cells. A major technical challenge is the culture of functional (perfused) networks. In this rapidly advancing field, some microfluidic devices are now getting close to the goal of an artificially perfused vascular network. Another development is the emergence of the zebrafish as a complementary model to mammals. In this review, we discuss the culture of endothelial cells and vascular networks from mammalian cells, and examine the prospects for using zebrafish cells for this objective. We also look into the future and consider how vascular networks in vitro might be successfully perfused using microfluidic technology. Show less
A major limitation to culturing tissues and organs is the lack of a functional vascular network in vitro. The zebrafish possess many useful properties which makes it a promising model for such... Show moreA major limitation to culturing tissues and organs is the lack of a functional vascular network in vitro. The zebrafish possess many useful properties which makes it a promising model for such studies. Unfortunately, methods of culturing endothelial cells from this species are not well characterised. Here, we tried two methods (embryoid body culture and organ explants from transgenic zebrafish kdrl:GFP embryos) to develop in vitro vascular networks. In the kdrl:GFP line, endothelial cells expresses green fluorescent protein, which allows to track the vascular development in live cultures. We found that embryoid bodies showed significantly longer and wider branches of connected endothelial cells when grown in a microfluidic system than in static culture. Similarly, sprouting of kdrl:GFP+ cells from the tissue explants was observed in a 3D hydrogel matrix. This study is a step towards the development of zebrafish vascular networks in vitro. Show less
Aquatic and terrestrial habitats are heterogeneous by nature with respect to sound and light conditions. Fish may extract signals and exploit cues from both ambient modalities and they may also... Show moreAquatic and terrestrial habitats are heterogeneous by nature with respect to sound and light conditions. Fish may extract signals and exploit cues from both ambient modalities and they may also select their sound and light level of preference in free-ranging conditions. In recent decades, human activities in or near water have altered natural soundscapes and caused nocturnal light pollution to become more widespread. Artificial sound and light may cause anxiety, deterrence, disturbance or masking, but few studies have addressed in any detail how fishes respond to spatial variation in these two modalities. Here we investigated whether sound and light affected spatial distribution and swimming behavior of individual zebrafish that had a choice between two fish tanks: a treatment tank and a quiet and light escape tank. The treatments concerned a 2 × 2 design with noisy or quiet conditions and dim or bright light. Sound and light treatments did not induce spatial preferences for the treatment or escape tank, but caused various behavioral changes in both spatial distribution and swimming behavior within the treatment tank. Sound exposure led to more freezing and less time spent near the active speaker. Dim light conditions led to a lower number of crossings, more time spent in the upper layer and less time spent close to the tube for crossing. No interactions were found between sound and light conditions. This study highlights the potential relevance for studying multiple modalities when investigating fish behavior and further studies are needed to investigate whether similar patterns can be found for fish behavior in free-ranging conditions. Show less
