Improved isolation of murine hepatocytes for in vitro malaria liver stage studies

<p>Abstract</p> <p>Background</p> <p>Primary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This paper describes an improved perfusion/dissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity.</p> <p>Results</p> <p>This protocol is a detailed description of a technique to isolate and culture mouse hepatocytes and represents an improvement over previous descriptions of hepatocyte isolation for malaria studies, regarding three technical aspects: (1) dissociation reagents choice; (2) cell separation gradient and (3) cell purity control. Cell dissociation was optimized for a specific collagenase digestion media. The cell dissociation step was improved by using a three-layer discontinuous gradient. A cell purity check was introduced to monitor the expression of CD95 on hepatocytes using flow cytometry methods.</p> <p>Conclusion</p> <p>The procedure described allows reproducible recovery of one to three million hepatocytes per preparation with cell purity of about 90% as determined by FACS analysis. Completion of the protocol is usually achieved in about four hours per preparation and pooling is suggested for multiple preparations of larger number of cells.</p

Accumulation of Plasmodium berghei-infected red blood cells in the brain is crucial for the development of cerebral malaria in mice

Copyright © 2010, American Society for Microbiology. All Rights Reserved.Cerebral malaria is the most severe complication of human infection with Plasmodium falciparum. It was shown that Plasmodium berghei ANKA-induced cerebral malaria was prevented in 100% of mice depleted of CD8+ T cells 1 day prior to the development of neurological signs. However, the importance of parasites in the brains of these mice was never clearly investigated. Moreover, the relevance of this model to human cerebral malaria has been questioned many times, especially concerning the relative importance of leukocytes versus parasitized erythrocytes sequestered in the brain. Here, we show that mice protected from cerebral malaria by CD8+ T-cell depletion have significantly fewer parasites in the brain. Treatment of infected mice with an antimalarial drug 15 to 20 h prior to the estimated time of death also protected mice from cerebral malaria without altering the number of CD8+ T cells in the brain. These mice subsequently developed cerebral malaria with parasitized red blood cells in the brain. Our results clearly demonstrated that sequestration of CD8+ T cells in the brain is not sufficient for the development of cerebral malaria in C57BL/6 mice but that the concomitant presence of parasitized red blood cells is crucial for the onset of pathology. Importantly, these results also demonstrated that the experimental cerebral malaria model shares many features with human pathology and might be a relevant model to study its pathogenesis.Fundação para a Ciência e a Tecnologia (FCT) with the coparticipation of FEDER, Portugal (POCTI/MGI/ 46719/2002). F. G. Baptista was supported by IEFP, Portugal, Ana C. Pena by grant IMM/BI/47-2008, and Ana Pamplona by grant SFRH/ BPD/26633/2006, both from FCT, Portugal

Chemokine Receptor CCR2 Is Not Essential for the Development of Experimental Cerebral Malaria

Infection with Plasmodium berghei ANKA induces cerebral malaria in susceptible mice. Brain-sequestered CD8(+) T cells are responsible for this pathology. We have evaluated the role of CCR2, a chemokine receptor expressed on CD8(+) T cells. Infected CCR2-deficient mice were as susceptible to cerebral malaria as wild-type mice were, and CD8(+) T-cell migration to the brain was not abolished

Species-Specific Inhibition of Cerebral Malaria in Mice Coinfected with Plasmodium spp.

Recent epidemiological observations suggest that clinical evolution of Plasmodium falciparum infections might be influenced by the concurrent presence of another Plasmodium species, and such mixed-species infections are now known to occur frequently in residents of most areas of endemicity. We used mice infected with P. berghei ANKA (PbA), a model for cerebral malaria (CM), to investigate the influence of experimental mixed-species infections on the expression of this pathology. Remarkably, the development of CM was completely inhibited by the simultaneous presence of P. yoelii yoelii but not that of P. vinckei or another line of P. berghei. In the protected coinfected mice, the accumulation of CD8(+) T cells in the brain vasculature, a pivotal step in CM pathogenesis, was found to be abolished. Protection from CM was further found to be associated with species-specific suppression of PbA multiplication. These observations establish the concept of mixed Plasmodium species infections as potential modulators of pathology and open novel avenues to investigate mechanisms implicated in the pathogenesis of malaria

A investigação em fonologia do português Research in Portuguese phonology

Neste artigo pretende-se traçar uma panorâmica dos estudos de fonologia realizados em Portugal, com especial incidência nas análises formalizadas que se desenvolveram a partir da obra de Chomsky e Halle (1968). Na Introdução referem-se obras que marcaram a fonologia do Português Europeu antes dessa data, e apresentam-se os principais trabalhos de carácter dialectal e filológico, os estudos fonéticos e as obras que se integram na linguística estrutural. Na apresentação das análises formais distingue-se a fonologia generativa clássica das teorias que lhe sucederam O artigo tem um anexo que contém a bibliografia exaustiva dos livros e artigos publicados em Portugal a partir dos anos 70.<br>This paper is an overview of the phonological studies in Portuguese starting with the formal analyses developed in Portugal after the publication of The Sound Pattern of English (1968). The relevant works on European Portuguese published before Chomsky & Halle are included in the Introduction: the most important dialectal and philological works, phonetic studies as well as structural descriptions. Formal analyses are divided in two parts: those that follow standard generative phonology and those oriented by subsequent theories. The annex includes a comprehensive bibliography of all phonological books and papers published in Portugal after the seventies