Anja Jensen

University of Copenhagen · Centre for Medical Parasitology

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...

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) an...

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...

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 abi...

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...

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...

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 d...

Plasmodium falciparum, the deadliest form of human malaria, remains one of the major threats to human health in endemic regions. Its virulence is attributed to its ability to modify infected red blood cells (iRBC) to adhere to endothelial receptors by placing variable antigens known as PfEMP1 on the iRBC surface. PfEMP1 expression determines the cy...

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...

Plasmodium falciparum, the deadliest form of human malaria, remains one of the major threats to human health in endemic regions. Its virulence is attributed to its ability to modify infected red blood cells (iRBC) to adhere to endothelial receptors by placing variable antigens known as PfEMP1 on the surface of the red cell. PfEMP1 expression on the...

We have previously shown that conformational change in the β2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8⁺ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of...

Cerebral malaria (CM) is caused by the binding of Plasmodium falciparum–infected erythrocytes (IEs) to the brain microvasculature, leading to inflammation, vessel occlusion, and cerebral swelling. We have previously linked dual intercellular adhesion molecule-1 (ICAM-1)– and endothelial protein C receptor (EPCR)–binding P. falciparum parasites to t...

We have previously shown that beta 2 -integrin conformational change is a very early activation marker that can be detected with fluorescent multimers of its ligand ICAM-1 for a rapid assessment of antigen-specific CD8 ⁺ T cells. Here, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4 ⁺ T cells...

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...

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant sub-families. ICAM-1-binding Group A PfEMP1 proteins also bind EPCR and have been associated with cerebral malaria in children. IgG to these PfEMP1 is acquired later in life than to Group...

Efficient T cell responses require the firm adhesion of T cells to their targets, e.g., virus-infected cells, which depends on T cell receptor (TCR)–mediated activation of β 2 -integrins. Gα s -coupled receptor agonists are known to have immunosuppressive effects, but their impact on TCR-mediated integrin activation is unknown. Using multimers of p...

Erythrocyte Binding Antigen of 175 kDa (EBA-175) has a well-defined role in binding to glycophorin A (GpA) during Plasmodium falciparum invasion of erythrocytes. However, EBA-175 is shed post invasion and a role for this shed protein has not been defined. We show that EBA-175 shed from parasites promotes clustering of RBCs, and EBA-175-dependent cl...

The lack of suitable animal models for the study of cytoadhesion of P. falciparum-infected erythrocytes (IEs) has necessitated in vitro studies employing a range of cell lines of either human tumour origin (e.g., BeWo and C32 cells) or non-human origin (e.g., CHO cells). Of the human cells available, many were isolated from adults, or derived from...

Graphical summary to the article "Activated integrins identify functional antigen-specific CD8 + T cells within minutes after antigen stimulation": Following T-cell receptor-mediated stimulation, integrin activation occurs within seconds through a process known as "inside-out" signaling that leads to an affinity increase and clustering of membran...

Supplementary information to the article "Activated integrins identify functional antigen-specific CD8 + T cells within minutes after antigen stimulation"

Significance Assessing antigen-specific T cells is crucial for our understanding of immune reactions against pathogens and tumors, and for evaluating immunotherapies in patients. Existing techniques to evaluate the functionality of T lymphocytes all rely on de novo expression of proteins, typically intracellular cytokines, and therefore require ela...

Heterochromatin plays a central role in the process of immune evasion, pathogenesis, and transmission of the malaria parasite Plasmodium falciparum during blood stage infection. Here, we use ChIP sequencing to demonstrate that sporozoites from mosquito salivary glands expand heterochromatin at subtelomeric regions to silence blood-stage-specific ge...

Cerebral malaria (CM) is a potentially deadly outcome of Plasmodium falciparum malaria that is precipitated by sequestration of infected erythrocytes (IEs) in the brain. The adhesion of IEs to brain endothelial cells is mediated by a subtype of parasite-encoded PfEMP1 that facilitate dual binding to host ICAM-1 and EPCR. The PfEMP1 subtype is chara...

Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1 (ICAM-1), and endothelial protein C receptor (EPC...

Table S4. The ICAM-1-Binding Motif Is Commonly Found in P. falciparum Genomes, Related to Figures 2 and 3

Document S1. Supplemental Experimental Procedures, Figures S1–S7, and Tables S1–S3, S5, and S6

Table S7. Statistical Analysis of the Effect of Receptor and Shear Stress on Adhesion of HB3VAR03-, 3D7PFD1235w-, and IT4VAR13-Infected Erythrocytes under Physiological Flow Conditions and Primers Used for Amplification of PfEMP1-Specific DNA Sequences and Recombinant Proteins Produced, Related to Figures 1, 2, 3, 4, 5, and 6

Cerebral malaria is a deadly outcome of infection by Plasmodium falciparum, occurring when parasite-infected erythrocytes accumulate in the brain. These erythrocytes display parasite proteins of the PfEMP1 family that bind various endothelial receptors. Despite the importance of cerebral malaria, a binding phenotype linked to its symptoms has not b...

Background Malawi experienced prolonged use of sulfadoxine/pyrimethamine (SP) as the front-line anti-malarial drug, with early replacement of chloroquine and delayed introduction of artemisinin-based combination therapy. Extended use of SP, and its continued application in pregnancy is impacting the genomic variation of the Plasmodium falciparum po...

An efficient immune response requires a selective and strong interaction of T cells with endothelium or target cells. These interactions are strengthened by the rapid induction of beta2-integrin-mediated adhesion upon chemokine receptor or T-cell receptor (TCR) stimulation, respectively. We and others have shown that an increase in G-alpha-s-protei...

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mA...

Plasmodium falciparum causes the most severe form of malaria and is responsible for essentially all malaria-related deaths. The accumulation in various tissues of erythrocytes infected by mature P. falciparum parasites can lead to circulatory disturbances and inflammation, and is thought to be a central element in the pathogenesis of the disease. I...

The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected eryt...

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation re...

Background Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation. Methods We performed whole-genome sequencing of 69 P. falciparum isolates from Malawi and used population genetics approaches to investigate genetic di...

Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes. Additionally, ICAM-1 acts as receptor for pathogens li...

Cerebral Plasmodium falciparum malaria is characterized by adhesion of infected erythrocytes (IEs) to the cerebral microvasculature. This has been linked to parasites expressing the structurally related group A subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of IE adhesion ligands and to IEs with affinity for ICAM-1. Howe...

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time du...

How immunity to malaria develops remains one of the great unresolved issues in bio-medicine and resolution of its various paradoxes is likely to be the key to developing effective malaria vaccines. The basic epidemiological observations are; under conditions of intense natural transmission, humans do become immune to P. falciparum malaria, but this...

Table 1. Primers used for amplification of DNA encoding PFD1235w domains. The table provides information on primers used for PCR amplification of amplicons encoding different PFD1235w protein domains.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60 different PfEMP1 variants, each PfEMP1 comprises several d...

The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherich...

PfEMP1 surface expression on erythrocytes infected with 3D7PFD1235w/PF11_0008 clone 3 and 4. Single colour surface staining of PfEMP1 by flow cytometry was done using (A) rabbit pre-bleed (1), αPFD1235w-DBL4γ (2), αPF11_0008-CIDR2β (3) antisera, and (B) rat pre-bleed (1), αPFD1235wDBL4 γ (2), αPFD1235w-DBL5δ-CIDR2β (3), and αPF11_0008-DBL4β (4) ant...

The specificity of antisera and antibodies used for surface labelling and selection of parasites. The specificity was tested by ELISA (A-C) and by Luminex (D-F) on 49 different PfEMP1 domains (Table S1). (A) rat PFD1235w-DBL4γ antisera, (B) rabbit PF11_0008-CIDR2β antisera, (C) rabbit FD1235w-CIDR1α antisera, (D) antibodies purified on a PFD1235w-D...

Simultaneous surface expression of PfEMP1 on single erythrocytes infected with 3D7 detected using purified antibodies. Parasite cultures (A-D) were identical to those used in Figure 1, Figure 2, and Figure S1. The affinity purified and depleted antibodies used were (A-D1) rat αPFD1235w-DBL4γ; (A-D2) rabbit PF11_0008-CIDR2β antibodies; and (A-D3), r...

Real-time Q-PCR Ct-values for exon I and intron crossing amplicons amplified from cDNA of 3D7. (A) 3D7PFD1235w sub-line. (B) 3D7PF11_0008 sub-line. (C) 3D7PFD1235w/PF11_0008 sub-line. Numbers on the Y-axis are primer numbers (See Table S2). Grey bars: exon I primers. Black bars: intron primers. White bars: house keeping gene primers. (0.01 MB PDF)

Simultaneous surface expression of PfEMP1 on single erythrocytes infected with Plasmodium falciparum 3D7PFD1235w/PF11_0008. Staining of PFD1235w and PF11_0008 is green and red, respectively. DAPI staining of DNA in the nuclei is blue. Scale bar 5 μm. (2.30 MB MOV)

Trypsination of 3D7 infected erythrocytes and blocking of antisera show no cross-reactivity. (A) and (D) 3D7PFD1235w IE, (B) and (E) 3D7PF11_0008 IE, (C) and (F) 3D7PFD1235w/Pf11_0008 IE. (A-C) Untreated (light grey bars), trypsin (black bars), and trypsin plus foetal calf serum (FCS) treated IE (dark grey bars) were surface stained for flow cytome...

Primers used for amplification of DNA encoding recombinant proteins coupled to Luminex beads. (0.09 MB DOC)

Specific var gene primers used for var intron Q-RT PCR. (0.05 MB DOC)

PfEMP1 surface expression on 3D7 infected erythrocytes and antisera specificity. (A) erythrocytes infected with the 3D7PFD1235w, (B) 3D7PF11_0008, and (C) 3D7PFD1235w/PF11_0008 sub-line. Surface staining of PfEMP1 by flow cytometry was done using rat antisera against (1) DBL1-CIDR1α, (2) CIDR1α*, (4) DBL3β, (5) DBL4γ, (7) DBL5δ and (8) DBL5δ-CIDR2β...

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured...

The binding of erythrocytes infected with mature blood stage parasites to the vascular bed is key to the pathogenesis of malignant malaria. The binding is mediated by members of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1s can be divided into groups, and it has previously been suggested that parasites expressing gro...

A subset of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1(SM)) is involved in the cytoadherence of P. falciparum-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1(SM) are encoded by group A var genes that are composed of a more constrained...

The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the prob...

Specific PfEMP1 domain primers used for PCR. Primers used to specifically amplify the PfEMP1 encoded fragments, the restriction enzyme sites are underlined and the extra bases precede the restriction sites.

The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythro...

In malaria endemic areas children may recover from malaria after chemotherapy in spite of harbouring genotypically drug-resistant Plasmodium falciparum. This phenomenon suggests that there is a synergy between drug treatment and acquired immunity. This hypothesis was examined in an area of moderately intense transmission of P. falciparum in Tanzani...

Variant surface antigens (VSA) on the surface of Plasmodium falciparum-infected red blood cells play a major role in the pathogenesis of malaria and are key targets for acquired immunity. The best-characterized VSA belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. In areas where P. falciparum is endemic, parasites causing...

Protection against Plasmodium falciparum malaria is largely mediated by IgG against surface Ags such as the erythrocyte membrane protein 1 family (PfEMP1) responsible for antigenic variation and sequestration of infected erythrocytes. PfEMP1 molecules can be divided into groups A, B/A, B, C, and B/C. We have previously suggested that expression of...

The var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is responsible for antigenic variation and sequestration of infected erythrocytes during malaria. We have previously grouped the 60 PfEMP1 variants of P. falciparum clone 3D7 into groups A and B/A (category A) and groups B, B/C, and C (category non-A). Express...