BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results.

Azithromycin disrupts apicoplast biogenesis in replicating and dormant liver stages of the relapsing malaria parasites Plasmodium vivax and Plasmodium cynomolgi.

The control and elimination of malaria caused by Plasmodium vivax is hampered by the threat of relapsed infection resulting from the activation of dormant hepatic hypnozoites. Currently, only the 8-aminoquinolines, primaquine and tafenoquine, have been approved for the elimination of hypnozoites, although their use is hampered by potential toxicity. Therefore, an alternative radical curative drug that safely eliminates hypnozoites is a pressing need.

Transfection Models to Investigate -Type Dormant Liver Stage Parasites.

causes the second highest number of malaria morbidity and mortality cases in humans. Several biological traits of this parasite species, including the formation of dormant stages (hypnozoites) that persist inside the liver for prolonged periods of time, present an obstacle for intervention measures and create a barrier for the elimination of malaria. Research into the biology of hypnozoites requires efficient systems for parasite transmission, liver stage cultivation and genetic modification.

Antimalarial Agents Targeting Plasmodium falciparum Carbonic Anhydrase: Towards Artesunate Hybrid Compounds with Dual Mechanism of Action.

Malaria continues to be a major public health challenge worldwide and, as part of the global effort toward malaria eradication, plasmodium carbonic anhydrases (CAs) have recently been proposed as potential targets for malaria treatment. In this study, a series of eight hybrid compounds combining the Artesunate core with a sulfonamide moiety were synthesized and evaluated for their inhibition potency against the widely expressed human (h) CAs I, II and the isoform from P. falciparum (PfCA).

Epigenetically regulated RNA-binding proteins signify malaria hypnozoite dormancy.

Dormancy enables relapsing malaria parasites, such as Plasmodium vivax and cynomolgi, to survive unfavorable conditions. It is enabled by hypnozoites, parasites remaining quiescent inside hepatocytes before reactivating and establishing blood-stage infection. We integrate omics approaches to explore gene-regulatory mechanisms underlying hypnozoite dormancy.

Taxanes trigger cancer cell killing in vivo by inducing non-canonical T cell cytotoxicity.

Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner.

Transcriptional profiling of hepatocytes infected with the replicative form of the malaria parasite Plasmodium cynomolgi.

Background: The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. The transcriptional profiling of P. cynomolgi liver stages was previously reported and revealed many important biological features of the parasite but left out the host response to malaria infection.

Sterile protection against relapsing malaria with a single-shot vaccine.

Vaccine development for Plasmodium vivax, an important human relapsing malaria, is lagging behind. In the case of the most deadly human malaria P. falciparum, unprecedented high levels of protection have been obtained by immunization with live sporozoites under accompanying chemoprophylaxis, which prevents the onset of blood-stage malaria.

Uncovering the mode of action of engineered T cells in patient cancer organoids.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity.

Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Anti-relapse Compounds.

Malaria hypnozoites are dormant parasite stages that reside inside hepatocytes. Upon activation, these stages can resume growth, causing new episodes of blood stage malaria infection. This chapter describes a fast and sensitive protocol for the detection of bioluminescent (BL) hypnozoites in vitro.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12).

The catechol derivative RC-12 (WR 27653) () is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard -rhesus monkey () model, but in a small clinical trial, it had no efficacy against hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and liver-stage activity of and its metabolites. Compound had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the -desmethyl, combined -desmethyl/-desethyl, and -didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of in rhesus monkeys vs that seen in humans.

Toward an understanding of the chemical ecology of alternative reproductive tactics in the bulb mite (Rhizoglyphus robini).

Background: Under strong sexual selection, certain species evolve distinct intrasexual, alternative reproductive tactics (ARTs). In many cases, ARTs can be viewed as environmentally-cued threshold traits, such that ARTs coexist if their relative fitness alternates over the environmental cue gradient. Surprisingly, the chemical ecology of ARTs has been underexplored in this context.

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite.

Novel bis-1,2,4-triazine compounds with potent activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites and .

Modeling Relapsing Malaria: Emerging Technologies to Study Parasite-Host Interactions in the Liver.

Recent studies of liver stage malaria parasite-host interactions have provided exciting new insights on the cross-talk between parasite and its mammalian (predominantly rodent) host. We review the latest state of the art and and zoom in on new technologies that will provide the tools necessary to investigate host-parasite interactions of relapsing parasites. Interactions between hypnozoites and hepatocytes are particularly interesting because the parasite can remain in a quiescent state for prolonged periods of time and triggers for reactivation have not been irrefutably identified.

A novel Axin2 knock-in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells.

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β-catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues.

Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Antirelapse Compounds.

Efforts to eradicate malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic parasite line that contains Nanoluc driven by the constitutive promoter, as well as firefly luciferase driven by the schizont-specific promoter.

A dual fluorescent reporter line reveals in vitro malaria hypnozoite reactivation.

malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P.

Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite during progression into dormancy.

Relapses of dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al.

A comparative transcriptomic analysis of replicating and dormant liver stages of the relapsing malaria parasite .

liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite .

A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P.

Antimalarial efficacy of MMV390048, an inhibitor of phosphatidylinositol 4-kinase.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all life cycle stages, apart from late hypnozoites in the liver.

Plasmodium knowlesi: a relevant, versatile experimental malaria model.

The primate malaria Plasmodium knowlesi has a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite-host interactions. The adaptation to long-term in vitro continuous blood stage culture in rhesus monkey, Macaca fascicularis and human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model.

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase.