Simple, sensitive and quantitative bioluminescence assay for determination of malaria pre-patent period

© 2014 Zuzarte-Luis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stateBackground: The first phase of malaria infection occurs in the liver and is clinically silent. Inside hepatocytes each Plasmodium sporozoite replicate into thousands of erythrocyte-infectious merozoites that when released into the blood stream result in clinical symptoms of the disease. The time between sporozoite inoculation and the appearance of parasites in the blood is defined as the pre-patent period, which is classically analysed by time-consuming and labor-intensive techniques, such as microscopy and PCR. Methods: Luciferase-expressing Plasmodium berghei parasites were used to measure pre-patent period of malaria infection in rodents using a bioluminescence assay that requires only one microliter of blood collected from the tail-vein. The accuracy and sensitivity of this new method was compared with conventional microscopy and PCR based techniques, and its capacity to measure the impact of anti-malarial interventions against the liver evaluated. Results: The described method is very sensitive allowing the detection of parasites during the first cycles of blood stage replication. It accurately translates differences in liver load due to inoculation of different sporozoite doses as well as a result of treatment with different primaquine regimens. Conclusions: A novel, simple, fast, and sensitive method to measure pre-patent period of malaria infection in rodents is described here. The sensitivity and accuracy of this new method is comparable to standard PCR and microscopy-based techniques, respectively.This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) grants PTDC/SAU-MIC/113697/2009 (VZL) and EXCL/IMI-MIC/0056/2012 (MMM).info:eu-repo/semantics/publishedVersio

Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis

Iron homeostasis is an essential biological process that ensures the tissue distribution of iron for various cellular processes. As the major producer of hepcidin, the liver is central to the regulation of iron metabolism. The liver is also home to many immune cells, which upon activation may greatly impact iron metabolism. Here, we focus on the role of invariant natural killer T (iNKT) cells, a subset of T lymphocytes that, in mice, is most abundant in the liver. Activation of iNKT cells with the prototypical glycosphingolipid antigen, α-galactosylceramide, resulted in immune cell proliferation and biphasic changes in iron metabolism. This involved an early phase characterized by hypoferremia, hepcidin induction and ferroportin suppression, and a second phase associated with strong suppression of hepcidin despite elevated levels of circulating and tissue iron. We further show that these changes in iron metabolism are fully dependent on iNKT cell activation. Finally, we demonstrate that the biphasic regulation of hepcidin is independent of NK and Kupfer cells, and is initially driven by the STAT3 infammatory pathway, whereas the second phase is regulated by repression of the BMP/SMAD signaling pathway. These fndings indicate that iNKT activation and the resulting cell proliferation infuence iron homeostasis

Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/.Iron homeostasis is an essential biological process that ensures the tissue distribution of iron for various cellular processes. As the major producer of hepcidin, the liver is central to the regulation of iron metabolism. The liver is also home to many immune cells, which upon activation may greatly impact iron metabolism. Here, we focus on the role of invariant natural killer T (iNKT) cells, a subset of T lymphocytes that, in mice, is most abundant in the liver. Activation of iNKT cells with the prototypical glycosphingolipid antigen, α-galactosylceramide, resulted in immune cell proliferation and biphasic changes in iron metabolism. This involved an early phase characterized by hypoferremia, hepcidin induction and ferroportin suppression, and a second phase associated with strong suppression of hepcidin despite elevated levels of circulating and tissue iron. We further show that these changes in iron metabolism are fully dependent on iNKT cell activation. Finally, we demonstrate that the biphasic regulation of hepcidin is independent of NK and Kupffer cells, and is initially driven by the STAT3 inflammatory pathway, whereas the second phase is regulated by repression of the BMP/SMAD signaling pathway. These findings indicate that iNKT activation and the resulting cell proliferation influence iron homeostasis.This work was supported by grants from the Canadian Institutes of Health Research (CIHR, Grant no. PJT-159775) and Natural Sciences and Engineering Research Council of Canada (NSERC, Grant RGPIN-2018-06442) to MMS. HH received a PhD scholarship from the NSERC. SL is a Research Scholars Emeritus awardee from the FRQS.info:eu-repo/semantics/publishedVersio

Host Cell Phosphatidylcholine Is a Key Mediator of Malaria Parasite Survival during Liver Stage Infection

During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.Seventh Framework Programme (European Commission) (Grant Agreement 311502)Fundacao para a Ciencia e a Tecnologia (Grant EXCL/IMI-MIC/0056/2012)Fundacao para a Ciencia e a Tecnologia (Grant PTDC/IMI-MIC/1568/2012

Active APPL1 sequestration by Plasmodium favors liver-stage development

© 2022 The Authors. This is an open access article under the CC BY-NC-ND license http://creativecommons.org/licenses/by-nc-nd/4.0/).Intracellular pathogens manipulate host cells to survive and thrive. Cellular sensing and signaling pathways are among the key host machineries deregulated to favor infection. In this study, we show that liver-stage Plasmodium parasites compete with the host to sequester a host endosomal-adaptor protein (APPL1) known to regulate signaling in response to endocytosis. The enrichment of APPL1 at the parasitophorous vacuole membrane (PVM) involves an atypical Plasmodium Rab5 isoform (Rab5b). Depletion of host APPL1 alters neither the infection nor parasite development; however, upon overexpression of a GTPase-deficient host Rab5 mutant (hRab5_Q79L), the parasites are smaller and their PVM is stripped of APPL1. Infection with the GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) in this case rescues the PVM APPL1 signal and parasite size. In summary, we observe a robust correlation between the level of APPL1 retention at the PVM and parasite size during exoerythrocytic development.This work was supported by grants from the la Caixa Banking Foundation and Fundação para a Ciência e a Tecnologia (HR17-00264 and PTDC/SAU-PAR/30751/2017 respectively, both to M.M.M.). A.L and S.S.B were sponsored by Fundação para a Ciência e a Tecnologia fellowships (PD/BD/114036/2015, SFRH/BD/114464/2016, respectively). V.S acknowledges funding from the Science and Engineering Research Board (SERB) (EMR/2016/006810), Department of Science and Technology (DST), Government of India. V.P. and C.S. were supported by GRK2581 (P6) SPHINGOINF of the Growing Spine Foundation (DFG).info:eu-repo/semantics/publishedVersio

Academic labs supporting COVID‐19 diagnostics

© 2021 Wiley-VCH GmbHDespite news about the new virus SARS-CoV-2 and COVID-19 grabbing the headlines in the beginning of 2020, life and work carried on as normal in most academic labs. Lab projects were the focus, results discussed face-to-face and hypothesis dismissed. Conferences were attended in person and flights to the next meeting often booked already. By March, the seriousness became clear. With surrounding countries reporting increasing number of infections, the emergence of SARS-CoV-2 in Portugal was a matter of time, with the first two Portuguese cases reported at the start of March. Newspapers reported the cases with warnings not to be alarmist and that officials would calmly follow the evolution of the events (see here). However, how do you follow the spread of a virus, especially when it is new and capacity to detect it is limited?We like to acknowledge the funding from the European Union H2020 ERA project (No 667824 – EXCELLtoINNOV) and the Fundação para a Ciência e a Tecnologia (FCT) research4COVID19 (n° 231_596873172, Generating SARS-CoV2 seroconversion assay: n° 162_596842560, Test, Test, Test: Diagnostic of COVID-19 and n° 729, High-throughput SARS-CoV2 neutralising antibodies assessment).info:eu-repo/semantics/publishedVersio

Parasite sensing of host nutrients and environmental cues

© 2018 Elsevier Inc.Parasites undergo complex life cycles that comprise a wide variety of cellular differentiation events in different host compartments and transmission across multiple hosts. As parasites depend on host resources, it is not surprising they have developed efficient mechanisms to sense alterations and adapt to the available resources in a wide range of environments. Here we provide an overview of the nutritional needs of different parasites throughout their diverse life stages and highlight recent insights into strategies that both hosts and parasites have developed to meet these nutritional requirements needed for defense, survival, and replication. These studies will provide the foundation for a systems-level understanding of host-parasite interactions, which will require the integration of molecular, epidemiologic, and mechanistic data and the application of interdisciplinary approaches to model parasite regulatory networks that are triggered by alterations in host resources.info:eu-repo/semantics/publishedVersio

Malaria infections: what and how can mice teach us

© Elsevier B.V. All rights reserved.Malaria imposes a horrific public health burden - hundreds of millions of infections and millions of deaths - on large parts of the world. While this unacceptable health burden and its economic and social impact have made it a focal point of the international development agenda, it became consensual that malaria control or elimination will be difficult to attain prior to gain a better understanding of the complex interactions occurring between its main players: Plasmodium, the causative agent of disease, and its hosts. Practical and ethical limitations exist regarding the ability to carry out research with human subjects or with human samples. In this review, we highlight how rodent models of infection have contributed significantly during the past decades to a better understanding of the basic biology of the parasite, host response and pathogenesis.V.Z.-L. is funded by FCT fellowship (SFRH/BPD/81953/2011).info:eu-repo/semantics/publishedVersio

Infection by Plasmodium changes shape and stiffness of hepatic cells

© 2012 Elsevier Inc. All rights reserved.Infection of liver cells by Plasmodium, the malaria parasite, is a clinically silent, obligatory step of the parasite's life cycle. The authors studied the progression of Plasmodium infection in hepatic cells by atomic force microscopy, measuring both topographical and nanomechanical changes upon infection. In recent years, several studies have suggested that cellular nanomechanical properties can be correlated with disease progression. The authors' results show that infected cells exhibit considerable topographical changes, which can be correlated with the presence of the parasite, leading to a significant roughening of the cell membrane. The nanomechanical measurements showed that infected cells were significantly stiffer than noninfected cells. Furthermore, the stiffening of the cells appeared to be a cellular reaction to the Plasmodium infection, rather than a result of the stiffness of the invading parasites themselves. This article provides the first evidence of mechanical changes occurring in hepatic cells in response to Plasmodium infection