Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

<p>Abstract</p> <p>Background</p> <p>Malaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the <it>ex-vivo </it>and <it>in-vitro </it>detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity.</p> <p>Methods</p> <p>A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and <it>Plasmodium falciparum </it>Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with <it>Plasmodium berghei </it>ANKA or <it>P. berghei </it>NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an <it>in-vitro </it>culture incubated with chloroquine or quinine.</p> <p>Results</p> <p>A flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from <it>in-vitro </it>experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively.</p> <p>Conclusions</p> <p>A simple modification of a flow cytometer allows for rapid and reliable detection and quantification of Hz-containing leukocytes and the analysis of differential surface marker expression in the same sample of Hz-containing <it>versus </it>non-Hz-containing leukocytes. Importantly, it distinguishes different maturation stages of parasitized RBC and may be the basis of a rapid no-added-reagent drug sensitivity assay.</p

Cerebral malaria and the hemolysis/methemoglobin/heme hypothesis: shedding new light on an old disease

Copyright © 2008 Elsevier Ltd. All rights reserved.Malaria causes more than 1 million deaths every year with cerebral malaria (CM) being a major cause of death in Sub-Saharan African children. The nature of the malaria-associated pathogenesis is complex and multi-factorial. A unified hypothesis involving sequestration of infected red blood cells, systemic host inflammatory response and hemostasis dysfunction has been proposed to explain the genesis of CM. In this review, we discuss the role of hemolysis, methemoglobin and free heme in CM, brought to light by our recent studies in mice as well as by other studies in humans.This work was supported by Fundação para a Ciência e Tecnologia, Portugal; Fellowships BPD/26633/2006 and BPD/31598/2006 to A.P. and S.E., respectively; Grant PTDC/SAU-MII/64125/2006 to A.P.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

Angiotensin II Is a New Component Involved in Splenic T Lymphocyte Responses during <i>Plasmodium berghei</i> ANKA Infection

<div><p>The contribution of T cells in severe malaria pathogenesis has been described. Here, we provide evidence for the potential role of angiotensin II (Ang II) in modulating splenic T cell responses in a rodent model of cerebral malaria. T cell activation induced by infection, determined by 3 to 4-fold enhancemen<b>t</b> in CD69 expression, was reduced to control levels when mice were treated with 20 mg/kg losartan (IC₅₀ = 0.966 mg/kg/d), an AT₁ receptor antagonist, or captopril (IC₅₀ = 1.940 mg/kg/d), an inhibitor of angiotensin-converting enzyme (ACE). Moreover, the production of interferon-γ and interleukin-17 by CD4⁺ T cells diminished 67% and 70%, respectively, by both treatments. Losartan reduced perforin expression in CD8⁺ T cells by 33% while captopril completely blocked it. The upregulation in chemokine receptor expression (CCR2 and CCR5) observed during infection was abolished and CD11a expression was partially reduced when mice were treated with drugs. T cells activated by <i>Plasmodium berghei</i> ANKA antigens showed 6-fold enhance in AT₁ levels in comparison with naive cells. The upregulation of AT₁ expression was reduced by losartan (80%) but not by captopril. Our results suggest that the AT₁/Ang II axis has a role in the establishment of an efficient T cell response in the spleen and therefore could participate in a misbalanced parasite-induced T cell immune response during <i>P. berghei</i> ANKA infection.</p></div