Cerebral malaria - modelling interactions at the blood-brain barrier in vitro.

The blood-brain barrier (BBB) is a continuous endothelial barrier that is supported by pericytes and astrocytes and regulates the passage of solutes between the bloodstream and the brain. This structure is called the neurovascular unit and serves to protect the brain from blood-borne disease-causing agents and other risk factors. In the past decade, great strides have been made to investigate the neurovascular unit for delivery of chemotherapeutics and for understanding how pathogens can circumvent the barrier, leading to severe and, at times, fatal complications. One such complication is cerebral malaria, in which Plasmodium falciparum-infected red blood cells disrupt the barrier function of the BBB, causing severe brain swelling. Multiple in vitro models of the BBB are available to investigate the mechanisms underlying the pathogenesis of cerebral malaria and other diseases. These range from single-cell monolayer cultures to multicellular BBB organoids and highly complex cerebral organoids. Here, we review the technologies available in malaria research to investigate the interaction between P. falciparum-infected red blood cells and the BBB, and discuss the advantages and disadvantages of each model.

Receptor Affinity-Based Purification of PfEMP1 Proteins.

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IEs) to bind a range of human receptors. This binding is mediated by a family of highly polymorphic proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are expressed on the surface of IEs and are composed of extracellular domains (NTS, CIDR, DBL), a transmembrane region and an acidic C-terminal segment. Subdomains of the extracellular N-terminal part of PfEMP1 molecules have been shown to bind specific receptors.In this chapter, we describe how to purify PfEMP1 proteins by a receptor affinity-based method. This includes how to prepare affinity columns and how to subsequently test the functionality of the purified PfEMP1 protein in an ELISA-based assay.

Affinity Purification of PfEMP1-Specific Antibodies from Human Blood.

Acquired immunity against Plasmodium falciparum infections relies heavily on IgG antibodies specific for PfEMP1 proteins expressed on the surface of infected erythrocytes. Purified human antibodies can be used, for example, to study the interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells, and to identify which IgG antibodies play a functional role in natural acquired immunity.This chapter describe how to affinity purify PfEMP1-specific human antibodies on an affinity column coupled with PfEMP1 protein. We include ELISA-based methods for identification of human plasma samples reactive against PfEMP1, and for testing of affinity purified IgG antibodies prior to their use in more advanced procedures.

Production of anti-PfEMP1 Polyclonal Antisera in Rats and Mice.

Plasmodium falciparum-infected erythrocytes (IEs) bind various host receptors via members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of the IEs. Antibody reagents are needed to investigate interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells. This protocol describes the production of rat and mouse polyclonal anti-PfEMP1 antibodies. Polyclonal antibodies are relatively easy to produce and have advantages compared to monoclonal antibodies (see Chapters 28 - 30 ) for some applications. An ELISA-based method to test the polyclonal antibodies before their use in more advanced procedures is also presented.

Analysis of Antibody Inhibition of PfEMP1 Binding by Competition ELISA.

Plasmodium falciparum express variant antigens on the surface of infected erythrocytes (IEs). These include proteins of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1 proteins mediate binding of IEs to various human endothelial receptors and induce antibodies response during natural infections. In this chapter, we describe a competition ELISA that is an assay for antibodies neutralizing binding of PfEMP1 proteins to their cognate receptor.

Chip-Based Assay of Adhesion of Plasmodium falciparum-Infected Erythrocytes to Cells Under Flow.

Unique to Plasmodium falciparum malaria parasites, the mature asexual stages of the life cycle are absent from the peripheral blood stream. Using syringe pumps and commercially available microslides, it is possible to mimic the blood flow, and investigate the interactions of erythrocytes infected by well-defined P. falciparum isolates for their ability to bind to various tissue receptors under physiological flow conditions. This chapter outlines the techniques needed to investigate how parasites bind to endothelial cells under physiological sheer stress conditions.

3D Organoid Assay of the Impact of Infected Erythrocyte Adhesion on the Blood-Brain Barrier.

Mass sequestration of Plasmodium falciparum parasites in the brain microvasculature can lead to cerebral malaria (CM), characterized by inflammation, vessel occlusion, and brain swelling. To date, only single-cell-type, monolayer assays have been used to investigate the effect of infected erythrocytes (IEs) on the human blood-brain barrier (BBB) and the underlying parenchyma. Here we present a human-derived 3D model of the BBB comprised of endothelial cells, pericytes, and astrocytes in direct contact with each other. The organoids readily self-assemble and can easily be grown in 96-well plates, allowing for high-throughput analysis. These organoids allow for the assessment of parasite adhesion, and analysis of barrier function, and gross morphological changes in response to parasite exposure.

IRDye800CW labeled uPAR-targeting peptide for fluorescence-guided glioblastoma surgery: Preclinical studies in orthotopic xenografts.

Glioblastoma (GBM) is a devastating cancer with basically no curative treatment. Even with aggressive treatment, the median survival is disappointing 14 months. Surgery remains the key treatment and the postoperative survival is determined by the extent of resection. Unfortunately, the invasive growth with irregular infiltrating margins complicates an optimal surgical resection. Precise intraoperative tumor visualization is therefore highly needed and molecular targeted near-infrared (NIR) fluorescence imaging potentially constitutes such a tool. The urokinase-type Plasminogen Activator Receptor (uPAR) is expressed in most solid cancers primarily at the invading front and the adjacent activated peritumoral stroma making it an attractive target for targeted fluorescence imaging. The purpose of this study was to develop and evaluate a new uPAR-targeted optical probe, IRDye800CW-AE344, for fluorescence guided surgery (FGS). Methods: In the present study we characterized the fluorescent probe with regard to binding affinity, optical properties, and plasma stability. Further, in vivo imaging characterization was performed in nude mice with orthotopic human patient derived glioblastoma xenografts, and we performed head-to-head comparison within FGS between our probe and the traditional procedure using 5-ALA. Finally, the blood-brain barrier (BBB) penetration was characterized in a 3D BBB spheroid model. Results: The probe effectively visualized GBM in vivo with a tumor-to-background ratio (TBR) above 4.5 between 1 to 12 h post injection and could be used for FGS of orthotopic human glioblastoma xenografts in mice where it was superior to 5-ALA. The probe showed a favorable safety profile with no evidence of any acute toxicity. Finally, the 3D BBB model showed uptake of the probe into the spheroids indicating that the probe crosses the BBB. Conclusion: IRDye800CW-AE344 is a promising uPAR-targeted optical probe for FGS and a candidate for translation into human use.

Cerebral Plasmodium falciparum malaria: The role of PfEMP1 in its pathogenesis and immunity, and PfEMP1-based vaccines to prevent it.

Malaria, a mosquito-borne infectious disease caused by parasites of the genus Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium-parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti-malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto-adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti-disease malaria vaccine. Here we review the role of ICAM-1 and EPCR receptor adhering falciparum-parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.