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Column: Out of sight, out of mind? How has imaging advanced our understanding of Plasmodium infected erythrocytes?

March 12, 2014 - 21:57 -- Bart G.J. Knols
Being a lab-based malaria researcher in a non-endemic country, I’ve been really interested by the columns that our colleagues ‘in the field’ have shared; looking at vector populations, elimination strategies and experiences of disease themselves. My perspectives of malaria are rather different however, and I hope to share more of a laboratory angle in my contributions to MalariaWorld this year. With that in mind, where better to start than the microscope and how most of us first come ‘face to face’ with Plasmodium spp? 
A bit of history
Much of our knowledge of Plasmodium parasites has come from the ability to visualize them (reviewed by [1]). The first description of the causative agents of malaria by Laveran in 1880 was made possible by 400x magnification of infected blood samples, and detected the pigment we now know as haemozoin. Since then the Giemsa stain, developed in 1904, has made it possible to differentiate between species, and remains the clinical gold standard to diagnose infection... 
This ability to selectively stain infected red blood cells (iRBCs) enabled their detection in tissues such as the brain post-mortem by Marchiafava, Bignami, and subsequently others (reviewed in [2]). These early studies proposed that iRBCs mechanically impaired blood flow as a result of “the accumulation in the cerebral vessels of red blood corpuscles loaded with amoebae”. Many theories have since been put forward to explain how severe complications could develop during malaria infection, yet this original concept of mechanical obstruction - arising from iRBC binding to vascular endothelial cells and/or downstream responses to this behaviour - remains a plausible mechanism. Other known features of severe malaria syndromes such as endothelial dysfunction and inflammatory processes may arise in subsequent responses to the initial iRBC binding event, however very little is known about how and when these processes occur during infection.
Examples of imaging approaches currently used to study iRBC behaviour
A variety of imaging methods have been applied to investigate cytoadhesion and other iRBC / host interactions that may contribute to development of complications during malaria infection. Some of these approaches are highlighted below to give an idea of how imaging may aid our understanding of iRBC-host interactions. To narrow this enormous field, here I will focus on examples of iRBC behaviours that could result in host pathology during clinical malaria and severe episodes:
Timelapse microscopy has enabled accurate analysis and timing of the invasion and egress processes, which are of crucial importance for understanding the erythrocytic growth cycle (for video click here). One exciting application of this technology has been to fluorescently label DNA-associated histone proteins and detect when these are released from parasites during schizont rupture [3]. The released histones stimulated human endothelial cells to produce inflammatory mediators and increase vascular permeability, which amongst other effects close to the original site of iRBC binding could cumulatively result in damage to the affected organ.
Microfluidic devices have permitted imaging of how iRBCs from different infections bind to vascular receptors and to uninfected RBCs under conditions that simulate blood flow [4]. For example, the following link shows iRBCs rolling within a commercially available biochip coated with intercellular adhesion molecule-1, which is commonly bound by iRBCs from patients with cerebral malaria (Cellix ltd., for video click here). 
The methodical characterisation of animal models has made it possible to directly visualize the microvasculature in vivo during controlled infections. A variety of intravital microscopy techniques have been used to investigate leukocyte and platelet recruitment, measurement of blood velocity, and many more factors during rodent malaria infection. To underscore how valuable these approaches are to our understanding of parasite and host interaction during malaria, Parasitology International has dedicated a recent issue to “in vivo imaging of parasite infection” (see here, pp 149-268). The placenta is a good example, where two-photon microscopy has been used to demonstrate that differences in the rate of blood flow encourage iRBC accumulation in areas with low flow rates, permitting stable binding of iRBCs to trophoblasts [5]. 
Direct in vivo analyses of human iRBC behaviours are difficult to undertake, however in recent years observation of the vasculature in the retina and mucosa have demonstrated that P. falciparum iRBC sequestration does occur in numerous sites in patients with cerebral malaria (reviewed by [2]). In addition, iRBC rolling along human arterioles and post-capillary venules has been demonstrated via an in vivo model system using human dermal blood vessels implanted into mice ([6]: Video 1). 
This ‘rolling’ behaviour of P. falciparum iRBCs has thus been visualised under flow conditions in different settings, suggesting that it could also occur during human infections. Unfortunately (besides ethical constraints!), the scale of imaging possible in patients is not amenable to answering mechanistic questions. However, if such behaviours could be detected in patients and related to risk of poor outcome, it may be possible to understand which molecular interactions novel therapies could best aim to interfere with. A clear example is iRBC rosetting (binding to uninfected RBCs), a behaviour strongly associated with pathogenicity. Binding of iRBCs to the host receptor heparan sulphate may be disrupted by depolymerized glycosaminoglycan [7], derived from heparin, which is currently under clinical testing as a potential adjunctive therapy. 
Seeing parasites can also help others to engage
Not only can imaging approaches provide novel insights into parasite behaviour, they can also make biological phenomena much easier to explain to non-scientists. I wonder how many malaria patients have actually seen what their infected blood looks like? Last month in the UK, the BBC screened a parasitology programme “Infested! Living With Parasites”, where Michael Mosely described a number of common parasites, which many of the British public may never have heard of. During the episode, Michael infected himself with a beef tapeworm, which was clearly visible in his gut via endoscopy (see here). Of particular interest for us as malaria professionals, he also observed P. falciparum infecting his own red blood cells (in the lab of Mike Blackman), where he was unnerved by the explosive egress of merozoites during schizont rupture. Programmes like this are useful for us as scientists to help others relate to our work, and importantly, might be one of the first encounters for younger viewers to be exposed to the fascinating behaviours of parasites, and which might spark a lifetime of interest!  
1. Cox FEG. 2010. History of the discovery of the malaria parasites and their vectors Parasites & Vectors. 3: 5.
2. White NJ, Turner GD, Day NP, Dondorp AM. 2013. Lethal malaria: Marchiafava and Bignami were right. J Infect Dis. 208: 192.
3. Gillrie MR, Lee K, Gowda DC, Davis SP, Monestier M, Cui L, Hien TT, Day NPJ, Ho M. 2012. Plasmodium falciparum Histones Induce Endothelial Proinflammatory Response and Barrier Dysfunction. Am J Pathol. 180: 1028. 
4. Herricks T, Seydel KB, Turner G, Molyneux M, Heyderman R, Taylor T, Rathod PK. 2011. A microfluidic system to study cytoadhesion of Plasmodium falciparum infected erythrocytes to primary brain microvascular endothelial cells. Lab Chip. 11: 2994.
5. de Moraes LV, Tadokoro CE, Gómez-Conde I, Olivieri DN, Penha-Gonçalves C. 2013. Intravital Placenta Imaging Reveals Microcirculatory Dynamics Impact on Sequestration and Phagocytosis of Plasmodium-Infected Erythrocytes. PLoS Pathog. 9: e1003154.
6. Ho M, Hickey MJ, Murray AG, Andonegui G, Kubes P. 2000. Visualization of Plasmodium falciparum-endothelium interactions in human microvasculature: mimicry of leukocyte recruitment. J. Exp. Med. 192: 1205.
7. Vogt AM, Pettersson F, Moll K, Jonsson C, Normark J, Ribacke U, Egwang TG, Ekre HP, Spillmann D, Chen Q, Wahlgren M. 2006. Release of sequestered malaria parasites upon injection of a glycosaminoglycan. PLoS Pathog. 2: e100.

Jenni Lawton is a post-doctoral researcher at the University of Glasgow, UK. Her research interests focus on the interactions between Plasmodium infected red blood cells (iRBCs) and the host; dynamic processes which are still incompletely understood. The behaviour of iRBCs may have important implications both in generating effective immune responses and in the escalation of some malaria infections towards severe complications. This will be her first foray into communications and she hopes to provide some interesting perspectives from the lab to the MalariaWorld community!



Submitted by Ricardo Ataide on

Great article Jenni. As with many aspects of live, what our eyes can see has a much higher impact on our perception of things than any other sense (except when our imagination takes over). One thing that usually surprises me is the amount of lab-based scientists that work with malaria and that fix and stain slides every single day, but have never actually made a wet-prep and just look at the parasite alive and kicking. I admit that I do it every now and then and always spend a few minutes marvelling at the sight of the parasite munching away inside the parasitophorous vacuole and the hemozoin spinning furiously!

Ricardo Ataíde