Bioprinted cancer model of neuroblastoma in a renal microenvironment as an efficiently applicable drug testing platform

Development of new anticancer drugs with currently available animal models is hampered by the fact that human cancer cells are embedded in an animal-derived environment. Neuroblastoma is the most common extracranial solid malignancy of childhood. Major obstacles include managing chemotherapy-resistant relapses and resistance to induction therapy, leading to early death in very-high-risk patients. Here, we present a three-dimensional (3D) model for neuroblastoma composed of IMR-32 cells with amplified genes of the myelocytomatosis viral related oncogene MYCN and the anaplastic lymphoma kinase (ALK) in a renal environment of exclusively human origin, made of human embryonic kidney 293 cells and primary human kidney fibroblasts. The model was produced with two pneumatic extrusion printheads using a commercially available bioprinter. Two drugs were exemplarily tested in this model: While the histone deacetylase inhibitor panobinostat selectively killed the cancer cells by apoptosis induction but did not affect renal cells in the therapeutically effective concentration range, the peptidyl nucleoside antibiotic blasticidin induced cell death in both cell types. Importantly, differences in sensitivity between two-dimensional (2D) and 3D cultures were cell-type specific, making the therapeutic window broader in the bioprinted model and demonstrating the value of studying anticancer drugs in human 3D models. Altogether, this cancer model allows testing cytotoxicity and tumor selectivity of new anticancer drugs, and the open scaffold design enables the free exchange of tumor and microenvironment by any cell type.DFG, 414044773, Open Access Publizieren 2021 - 2022 / Technische Universität Berli

Staphylococcus saccharolyticus Isolated From Blood Cultures and Prosthetic Joint Infections Exhibits Excessive Genome Decay

The slow-growing, anaerobic, coagulase-negative species Staphylococcus saccharolyticus is found on human skin and in clinical specimens but its pathogenic potential is unclear. Here, we investigated clinical isolates and sequenced the genomes of seven strains of S. saccharolyticus. Phylogenomic analyses showed that the closest relative of S. saccharolyticus is Staphylococcus capitis with an average nucleotide identity of 80%. Previously sequenced strains assigned to S. saccharolyticus are misclassified and belong to S. capitis. Based on single nucleotide polymorphisms of the core genome, the population of S. saccharolyticus can be divided into two clades that also differ in a few larger genomic islands as part of the flexible genome. An unexpected feature of S. saccharolyticus is extensive genome decay, with over 300 pseudogenes, indicating ongoing reductive evolution. Many genes of the core metabolism are not functional, rendering the species auxotrophic for several amino acids, which could explain its slow growth and need for fastidious growth conditions. Secreted proteins of S. saccharolyticus were determined; they include stress response proteins such as heat and oxidative stress-related factors, as well as immunodominant staphylococcal surface antigens and enzymes that can degrade host tissue components. The strains secrete lipases and a hyaluronic acid lyase. Hyaluronidase as well as urease activities were detected in biochemical assays, with clade-specific differences. Our study revealed that S. saccharolyticus has adapted its genome, possibly due to a recent change of habitat; moreover, the data imply that the species has tissue-invasive potential and might cause prosthetic joint infections

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen's elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

3D-bioprinted HepaRG cultures as a model for testing long term aflatoxin B1 toxicity in vitro

In recent years 3D-bioprinting technology has been developed as an alternative to animal testing. It possesses a great potential for in vitro testing as it aims to mimic human organs and physiology. In the present study, an alginate-gelatin-Matrigel based hydrogel was used to prepare 3D-bioprinted HepaRG cultures using a pneumatic extrusion printer. These 3D models were tested for viability and metabolic functions. Using 3D-bioprinted HepaRG cultures, we tested the toxicity of aflatoxin B1 (10 or 20 μM) in vitro and compared the results with 2D HepaRG cultures. There was a dose-dependent toxicity effect on cell viability, reduction of metabolic activity and albumin production. We found that 3D-bioprinted HepaRG cultures are more resistant to aflatoxin B1 treatment than 2D cultures. Although the metabolic activities were reduced upon treatment with aflatoxin B1, the 3D models were still viable and survived longer, up to 3 weeks, than the 2D culture, as visualized by fluorescence microscopy. Furthermore, albumin production recovered slightly in 3D models after one and two weeks of treatment. Taken together, we consider using 3D-bioprinting technology to generate 3D tissue models as an alternative way to study toxicity in vitro and this could also provide a suitable alternative for chronic hepatotoxicity studies in vitro.TU Berlin, Open-Access-Mittel – 202

Developmental Landscape of Potential Vaccine Candidates Based on Viral Vector for Prophylaxis of COVID-19

Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, arose at the end of 2019 as a zoonotic virus, which is the causative agent of the novel coronavirus outbreak COVID-19. Without any clear indications of abatement, the disease has become a major healthcare threat across the globe, owing to prolonged incubation period, high prevalence, and absence of existing drugs or vaccines. Development of COVID-19 vaccine is being considered as the most efficient strategy to curtail the ongoing pandemic. Following publication of genetic sequence of SARS-CoV-2, globally extensive research and development work has been in progress to develop a vaccine against the disease. The use of genetic engineering, recombinant technologies, and other computational tools has led to the expansion of several promising vaccine candidates. The range of technology platforms being evaluated, including virus-like particles, peptides, nucleic acid (DNA and RNA), recombinant proteins, inactivated virus, live attenuated viruses, and viral vectors (replicating and non-replicating) approaches, are striking features of the vaccine development strategies. Viral vectors, the next-generation vaccine platforms, provide a convenient method for delivering vaccine antigens into the host cell to induce antigenic proteins which can be tailored to arouse an assortment of immune responses, as evident from the success of smallpox vaccine and Ervebo vaccine against Ebola virus. As per the World Health Organization, till January 22, 2021, 14 viral vector vaccine candidates are under clinical development including 10 nonreplicating and four replicating types. Moreover, another 39 candidates based on viral vector platform are under preclinical evaluation. This review will outline the current developmental landscape and discuss issues that remain critical to the success or failure of viral vector vaccine candidates against COVID-19

Evaluating the Suitability of 3D Bioprinted Samples for Experimental Radiotherapy: A Pilot Study

Radiotherapy is an important component in the treatment of lung cancer, one of the mostcommon cancers worldwide, frequently resulting in death within only a few years of diagnosis. Inorder to evaluate new therapeutic approaches and compare their efficiency with regard to tumourcontrol at a pre-clinical stage, it is important to develop standardized samples which can serveas inter-institutional outcome controls, independent of differences in local technical parameters orspecific techniques. Recent developments in 3D bioprinting techniques could provide a sophisticatedsolution to this challenge. We have conducted a pilot project to evaluate the suitability of standardizedsamples generated from 3D printed human lung cancer cells in radiotherapy studies. The sampleswere irradiated at high dose rates using both broad beam and microbeam techniques. We found the3D printed constructs to be sufficiently mechanically stable for use in microbeam studies with peakdoses up to 400 Gy to test for cytotoxicity, DNA damage, and cancer cell death in vitro. The results ofthis study show how 3D structures generated from human lung cancer cells in an additive printingprocess can be used to study the effects of radiotherapy in a standardized manner

Chlamydia infection depends on a functional MdM2-p53 axis

Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection

Chlamydia infection depends on a functional MDM2-p53 axis.

Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection