Fatima El-Assaad

Platelet-endothelial cell interactions in cerebral malaria: The end of a cordial understanding

Cerebral malaria is an acute encephalopathy evolving from an infection with Plasmodium falciparum which kills more than one million people each year. Brain tissues from patients who died with cerebral malaria revealed multifocal capillary obstruction by parasitised red blood cells, platelets, and leukocytes. Many studies are unified in their proposal of two major hypotheses consisting of cell adhesion to the brain endothelium and excessive immune stimulation resulting in further vascular inflammation, prothrombotic cell activation, mechanical obstruction of cerebral capillaries and, consequently, blood-brain barrier disruption. Platelets and endothelial cells communicate on multiple levels. Infection-induced changes in platelets and endothelial cells occur in cerebral malaria, resulting in their concomitant activation, increased interactions between these two cell types, and a secondary procoagulant or hypercoagulable state. Here we review evidence for these mechanisms and highlight the possible role of platelets as effectors of endothelial damage in cerebral malaria. A better understanding of the complex regulation of these various interactions between brain endothelial cells and platelets in the context of cerebral malaria may prove useful in the development of new approaches to the treatment of this disease.

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

Platelet‐derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67‐labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker‐labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein‐loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca2+ chelation by EDTA, suggesting a partial role for receptor‐mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule‐1 and vascular cell adhesion molecule‐1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet‐derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin‐dependent phagocytosis. Platelet‐derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl‐β‐cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na+/H+ exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.

Real-time imaging reveals the dynamics of leukocyte behaviour during experimental cerebral malaria pathogenesis.

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6Chi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8+ T lymphocytes. A critical number of CD8+ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8+ T lymphocytes in regulating vascular pathology in this disease.

Production, Fate and Pathogenicity of Plasma Microparticles in Murine Cerebral Malaria

In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.

Cytoadherence of Plasmodium berghei-infected red blood cells to murine brain and lung microvascular endothelial cells in vitro.

ABSTRACT Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.

Differential microRNA expression in experimental cerebral and noncerebral malaria

ABSTRACT MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been implicated in the regulation of immune responses, but their role in the immune response to Plasmodium infection is unknown. We studied the expression of selected miRNAs following infection of CBA mice with Plasmodium berghei ANKA (PbA), which causes cerebral malaria (CM), or Plasmodium berghei K173 (PbK), which causes severe malaria but without cerebral complications, termed non-CM. The differential expression profiles of selected miRNAs (let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155) were analyzed in mouse brain and heart tissue by quantitative reverse transcription-PCR (qRT-PCR). We identified three miRNAs that were differentially expressed in the brain of PbA-infected CBA mice: let7i, miR-27a, and miR-150. In contrast, no miRNA changes were detected in the heart, an organ with no known pathology during acute malaria. To investigate the involvement of let-7i, miR-27a, and miR-150 in CM-resistant mice, we assessed the expression levels in gamma interferon knockout (IFN-γ−/−) mice on a C57BL/6 genetic background. The expression of let-7i, miR-27a, and miR-150 was unchanged in both wild-type (WT) and IFN-γ−/− mice following infection. Overexpression of these three miRNAs during PbA, but not PbK, infection in WT mice may be critical for the triggering of the neurological syndrome via regulation of their potential downstream targets. Our data suggest that in the CBA mouse at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

In vitro culture of Plasmodium berghei-ANKA maintains infectivity of mouse erythrocytes inducing cerebral malaria.

BackgroundInfection with Plasmodium berghei is a widely used model of murine malaria and a powerful tool for reverse genetic and pathogenesis studies. However, the efficacy of in vitro reinvasion of erythrocytes is generally low, limiting in vitro studies.MethodsPlasmodium berghei ANKA-infected blood obtained from a susceptible infected mouse was cultured in various conditions and in vitro parasitaemia was measured every day to evaluate the rate of reinvasion.ResultsHigh quality culture media were used and reinvasion rates were improved by vigorous orbital shaking of the flask and increasing density of the medium with gelatin.DiscussionUsing these settings, reinvasion of normal mouse erythrocytes by the parasite was obtained in vitro over two weeks with preservation of the infectivity in vivo.

Citicoline (CDP-choline): What role in the treatment of complications of infectious diseases

A dysregulated host immune response, as opposed to the intrinsic virulence of a microbial pathogen induces a large part of the pathology seen in infectious diseases. However, current therapies are designed to target the pathogen rather than the underlying pathogenic mechanisms responsible for the manifestation of the pathology. Recent studies have highlighted the role of endothelial cell alteration in the pathology induced in sepsis and cerebral malaria. The endothelial onslaught described, is similar to that seen during ischemia reperfusion in stroke. Protecting endothelial cell membranes during sepsis and cerebral malaria, using citicoline in the same way as in stroke, has thus emerged as a new strategy that needs to be evaluated urgently. Citicoline is a natural compound that is registered for use in ischemic stroke, head trauma and neurological disorders. It enters the phosphatidylcholine synthesis pathway as a rate-limiting step and is involved in the modulation of a large number of metabolic pathways and neurotransmitter levels, and also in the biosynthesis of phospholipids in neuronal membranes. This short review highlights the potential role of citicoline as part of adjunct therapy in the treatment of infectious diseases.

FTIR Imaging of Brain Tissue Reveals Crystalline Creatine Deposits Are an ex Vivo Marker of Localized Ischemia during Murine Cerebral Malaria: General Implications for Disease Neurochemistry

Phosphocreatine is a major cellular source of high energy phosphates, which is crucial to maintain cell viability under conditions of impaired metabolic states, such as decreased oxygen and energy availability (i.e., ischemia). Many methods exist for the bulk analysis of phosphocreatine and its dephosphorylated product creatine; however, no method exists to image the distribution of creatine or phosphocreatine at the cellular level. In this study, Fourier transform infrared (FTIR) spectroscopic imaging has revealed the ex vivo development of creatine microdeposits in situ in the brain region most affected by the disease, the cerebellum of cerebral malaria (CM) diseased mice; however, such deposits were also observed at significantly lower levels in the brains of control mice and mice with severe malaria. In addition, the number of deposits was observed to increase in a time-dependent manner during dehydration post tissue cutting. This challenges the hypotheses in recent reports of FTIR spectroscopic imaging where creatine microdeposits found in situ within thin sections from epileptic, Alzheimers (AD), and amlyoid lateral sclerosis (ALS) diseased brains were proposed to be disease specific markers and/or postulated to contribute to the brain pathogenesis. As such, a detailed investigation was undertaken, which has established that the creatine microdeposits exist as the highly soluble HCl salt or zwitterion and are an ex-vivo tissue processing artifact and, hence, have no effect on disease pathogenesis. They occur as a result of creatine crystallization during dehydration (i.e., air-drying) of thin sections of brain tissue. As ischemia and decreased aerobic (oxidative metabolism) are common to many brain disorders, regions of elevated creatine-to-phosphocreatine ratio are likely to promote crystal formation during tissue dehydration (due to the lower water solubility of creatine relative to phosphocreatine). The results of this study have demonstrated that although the deposits do not occur in vivo, and do not directly play any role in disease pathogenesis, increased levels of creatine deposits within air-dried tissue sections serve as a highly valuable marker for the identification of tissue regions with an altered metabolic status. In this study, the location of crystalline creatine deposits were used to identify whether an altered metabolic state exists within the molecular and granular layers of the cerebellum during CM, which complements the recent discovery of decreased oxygen availability in the brain during this disease.

Mechanisms of murine cerebral malaria: Multimodal imaging of altered cerebral metabolism and protein oxidation at hemorrhage sites

Multimodal spectroscopic imaging resolved controversies on biochemical changes associated with cerebral malaria pathology. Using a multimodal biospectroscopic approach, we settle several long-standing controversies over the molecular mechanisms that lead to brain damage in cerebral malaria, which is a major health concern in developing countries because of high levels of mortality and permanent brain damage. Our results provide the first conclusive evidence that important components of the pathology of cerebral malaria include peroxidative stress and protein oxidation within cerebellar gray matter, which are colocalized with elevated nonheme iron at the site of microhemorrhage. Such information could not be obtained previously from routine imaging methods, such as electron microscopy, fluorescence, and optical microscopy in combination with immunocytochemistry, or from bulk assays, where the level of spatial information is restricted to the minimum size of tissue that can be dissected. We describe the novel combination of chemical probe–free, multimodal imaging to quantify molecular markers of disturbed energy metabolism and peroxidative stress, which were used to provide new insights into understanding the pathogenesis of cerebral malaria. In addition to these mechanistic insights, the approach described acts as a template for the future use of multimodal biospectroscopy for understanding the molecular processes involved in a range of clinically important acute and chronic (neurodegenerative) brain diseases to improve treatment strategies.