Broad inhibition of plasmodium falciparum cytoadherence by (+)-epigallocatechin gallate

<p>Abstract</p> <p>Background</p> <p>The surface antigen P<it>f</it>EMP-1 is a key virulence factor of the human malaria parasite implicated in the cytoadherence of <it>Plasmodium falciparum </it>infected erythrocytes to a range of receptors on host endothelium. Among these host receptors, binding to ICAM-1 is related to cerebral malaria. The majority of the mortality in children with cerebral malaria is seen within 24 h of hospital admission despite the use of effective anti-parasite drugs, therefore, the development of adjunctive therapies is urgently needed.</p> <p>The polyphenolic compound (+)-epigallocatechin gallate ((+)-EGCG) has been previously evaluated for anti-adhesive properties using a small number of laboratory parasite isolates. Here, this property is further explored using a new panel of ICAM-1-binding patient isolates of <it>P. falciparum </it>to ascertain if (+)-EGCG might be effective as a broad spectrum inhibitor of ICAM-1-based cytoadherence.</p> <p>Methods</p> <p><it>Plasmodium falciparum </it>lines, including A4 and ItG as positive controls and nine new ICAM-1 binding patient isolates, were allowed to bind with ICAM-1-Fc protein under static assay conditions in the presence and absence of 50 μM (+)-EGCG. Adhesion levels of all the parasite strains were quantified by microscopy as the mean number of infected erythrocyte (IE) bound per mm²of surface area and statistical comparisons were made to demonstrate the effect of (+)-EGCG on the binding of various parasite variants to human ICAM-1.</p> <p>Results</p> <p>This study revealed that binding of patient isolates to ICAM-1 was reduced significantly with inhibition levels of 37% in patient isolate BC-12 up to a maximum of 80% in patient isolate 8146 at 50 μM (+)-EGCG.</p> <p>Conclusion</p> <p>Evaluation of the anti-adhesive property of (+)-EGCG against a new panel of ICAM-1-binding patient isolates of <it>P. falciparum </it>showed that this inhibitor, identified as potential mimic of the L43 loop of human ICAM-1, was effective at blocking cytoadherence.</p

An external sensing system in Plasmodium falciparum-infected erythrocytes

Additional file 4. ItG-pRBC were treated with 10 ng/ml TNF for 60, 30, 15 and 5 min and the cellular proteins were extracted and separated by SDS-PAGE, followed by Western blot probed with anti-HSP90 and AB4 antibodies. The 90kDa protein recognized by the two antibodies showed similar expression patterns

The Role of Animal Models for Research on Severe Malaria

In light of the recent controversies over the role of animal models for research into the development of new treatments for severe malaria, particularly cerebral disease, a group of scientists came together to discuss the relative merits of a range of animal models and their overlap with the complex clinical syndromes of human disease. While it was not possible to fully resolve differences over the utility of the Plasmodium berghei ANKA model of experimental cerebral malaria, the meeting did bring the two research communities closer together to identify further work to provide information needed to validate the model and revitalise the development of other animal models displaying features of human pathology. The driving force behind this was the desire to ensure better translation of experimental findings into effective treatments for severe malaria

Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function

Background: Sequestration and cytoadherence of Plasmodium falciparum-infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria. Methods: We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with P. falciparum material, recombinant PfEMP1 or lysates from IE, and measured barrier function by trans endothelial electrical resistance (TEER). A selection of PAR1 inhibitors was tested for their ability to reverse the P. falciparum and thrombin induced decrease in barrier function. Results: An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, PAR1 inhibitors did not rescue the barrier dysfunction after co-culture with IE lysate. Conclusions: The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism. These findings have implications for the design of adjunct therapies to reduce brain swelling in cerebral malaria

Marked elevation in plasma osteoprotegerin constitutes an early and consistent feature of cerebral malaria

Adherence of infected erythrocytes to vascular endothelium causes acute endothelial cell (EC) activation during Plasmodium falciparum infection. Consequently, proteins stored in Weibel-Palade (WP) bodies within EC are secreted into the plasma. Osteoprotegerin (OPG) binds to VWF and consequently is stored within WP bodies. Given the critical role of EC activation in the pathogenesis of severe malaria, we investigated plasma OPG levels in children with P. falciparum malaria. At presentation, plasma OPG levels were significantly elevated in children with cerebral malaria (CM) compared to healthy controls (means 16.0 vs 0.8 ng/ml; p<0.01). Importantly, OPG levels were also significantly higher in children with CM who had a fatal outcome, compared to children with CM who survived. Finally, in children with CM, plasma OPG levels correlated with other established prognostic indices (including plasma lactate levels and peripheral parasite density). To further investigate the relationship between severe malaria and OPG, we utilised a murine model of experimental CM in which C57BL/6J mice were infected with P. berghei ANKA. Interestingly, plasma OPG levels were increased 4.6 fold within 24 hours following P. berghei inoculation. This early marked elevation in OPG levels was observed before any objective clinical signs were apparent, and preceded the development of peripheral blood parasitaemia. As the mice became increasingly unwell, plasma OPG levels progressively increased. Collectively, these data suggest that OPG constitutes a novel biomarker with prognostic significance in patients with severe malaria. In addition, further studies are required to determine whether OPG plays a role in modulating malaria pathogenesis

HIV coinfection influences the inflammatory response but not the outcome of cerebral malaria in Malawian children

This work was supported by grants from the NIH (T.E.T., 5R01AI034969-14) and a Clinical Fellowship from The Wellcome Trust, United Kingdom (C.A.M, 88758). The Malawi-Liverpool-Wellcome Clinical Research Programme is supported by core funding from The Wellcome Trust, UK.Objectives. Study of the effect of HIV on disease progression in heterogeneous severe malaria syndromes with imprecise diagnostic criteria has led to varying results. Characteristic retinopathy refines cerebral malaria (CM) diagnosis, enabling more precise exploration of the hypothesis that HIV decreases the cytokine response in CM, leading to higher parasite density and a poor outcome. Methods. We retrospectively reviewed data on clinical progression and laboratory parameters in 877 retinopathy-positive CM cases admitted 1996-2011 (14.4% HIV-infected) to a large hospital in Malawi. Admission plasma levels of TNF, interleukin-10, and soluble intercellular adhesion molecule (sICAM-1) were measured by ELISA in 135 retinopathy-positive CM cases. Results. HIV-infected CM cases had lower median plasma levels of TNF (p=0.008), interleukin-10 (p=0.045) and sICAM-1 (p=0.04) than HIV-uninfected cases. Although HIV-infected children were older and more likely to have co-morbidities, HIV-status did not significantly affect parasite density (p=0.90) or outcome (24.8% infected, vs. 18.5% uninfected; p=0.13). Conclusions. In this well-characterised CM cohort, HIV-coinfection was associated with marked blunting of the inflammatory response but did not affect parasite density or outcome. These data highlight the complex influence of HIV on severe malaria and bring into question systemic inflammation as a primary driver of pathogenesis in human CM.Publisher PDFPeer reviewe

Malaria parasite tyrosyl-tRNA synthetase secretion triggers pro-inflammatory responses

Malaria infection triggers pro-inflammatory responses in humans that are detrimental to host health. Parasite-induced enhancement in cytokine levels correlate with malaria-associated pathologies. Here we show that parasite tyrosyl-tRNA synthetase (PfTyrRS), a housekeeping protein translation enzyme, induces pro-inflammatory responses from host immune cells. PfTyrRS exits from the parasite cytoplasm into the infected red blood cell (iRBC) cytoplasm, from where it is released into the extracellular medium on iRBC lysis. Using its ELR peptide motif, PfTyrRS specifically binds to and internalizes into host macrophages, leading to enhanced secretion of the pro-inflammatory cytokines TNF-α ± and IL-6. PfTyrRS-macrophage interaction also augments expression of adherence-linked host endothelial receptors ICAM-1 and VCAM-1. Our description of PfTyrRS as a parasite-secreted protein that triggers pro-inflammatory host responses, along with its atomic resolution crystal structure in complex with tyrosyl-adenylate, provides a novel platform for targeting PfTyrRS in anti-parasitic strategies

Cytoadherence and virulence - the case of Plasmodium knowlesi malaria

Background: Cytoadherence of infected red blood cells to brain endothelium is causally implicated in malarial coma, one of the severe manifestations of falciparum malaria. Cytoadherence is mediated by specific binding of variant parasite antigens, expressed on the surface of infected erythrocytes, to endothelial receptors including, ICAM-1, VCAM and CD36. In fatal cases of severe falciparum malaria with coma, blood vessels in the brain are characteristically congested with infected erythrocytes. Brain sections from a fatal case of knowlesi malaria, but without coma, were similarly congested with infected erythrocytes. The objective of this study was to determine the binding phenotype of Plasmodium knowlesi infected human erythrocytes to recombinant human ICAM-1, VCAM and CD36. Methods: Five patients with PCR-confirmed P. knowlesi malaria were recruited into the study with consent between April and August 2010. Pre-treatment venous blood was washed and cultured ex vivo to increase the proportion of schizont-infected erythrocytes. Cultured blood was seeded into Petri dishes with triplicate areas coated with ICAM-1, VCAM and CD36. Following incubation at 37°C for one hour the dishes were washed and the number of infected erythrocytes bound/mm2 to PBS control areas and to recombinant human ICAM-1 VCAM and CD36 coated areas were recorded. Each assay was performed in duplicate. Assay performance was monitored with the Plasmodium falciparum clone HB3. Results: Blood samples were cultured ex vivo for up to 14.5 h (mean 11.3 ± 1.9 h) to increase the relative proportion of mature trophozoite and schizont-infected red blood cells to at least 50% (mean 65.8 ± 17.51%). Three (60%) isolates bound significantly to ICAM-1 and VCAM, one (20%) isolate bound to VCAM and none of the five bound significantly to CD36. Conclusions: Plasmodium knowlesi infected erythrocytes from human subjects bind in a specific but variable manner to the inducible endothelial receptors ICAM-1 and VCAM. Binding to the constitutively-expressed endothelial receptor CD36 was not detected. Further work will be required to define the pathological consequences of these interactions

How Does Blood-Retinal Barrier Breakdown Relate to Death and Disability in Pediatric Cerebral Malaria?

Background In cerebral malaria, the retina can be used to understand disease pathogenesis. The mechanisms linking sequestration, brain swelling and death remain poorly understood. We hypothesized that retinal vascular leakage would be associated with brain swelling. Methods We used retinal angiography to study blood-retinal barrier integrity. We analyzed retinal leakage, histopathology, brain MRI, and associations with death and neurological disability in prospective cohorts of Malawian children with cerebral malaria. Results Three types of retinal leakage were seen: Large focal leak (LFL), punctate leak (PL) and vessel leak. LFL and PL were associated with death (OR 13.20, 95%CI 5.21-33.78 and 8.58, 2.56-29.08 respectively), and brain swelling (p<0.05). Vessel leak and macular non-perfusion were associated with neurological disability (3.71, 1.26-11.02 and 9.06, 1.79-45.90). LFL was observed as an evolving retinal hemorrhage. A core of fibrinogen and monocytes was found in 39 (93%) white-centered hemorrhages. Conclusions Blood-retina barrier breakdown occurs in three patterns in cerebral malaria. Associations between LFL, brain swelling, and death suggest that the rapid accumulation of cerebral hemorrhages, with accompanying fluid egress, may cause fatal brain swelling. Vessel leak from barrier dysfunction, and non-perfusion were not associated with severe brain swelling, but with neurological deficits, suggesting hypoxic injury in survivors