Rowe, Alex

MacCormick, Ian

Diallo, Nouhoum

University of Edinburgh

Darwin Trust of Edinburgh

2026-03-25T18:46:06Z

2026-03-25

Cerebral malaria (CM) is characterised by the adhesion and sequestration of Plasmodium falciparum infected erythrocytes (IEs) in the human brain microvascular endothelial cells (HBECs). IEs can adhere to immortalised human brain endothelial cells (hCMEC/D3) in vitro. This provides a reliable model for the investigation of interactions between IEs and human brain endothelium. Sequestration is mediated by interactions between members of the P. falciparum erythrocyte membrane protein 1(PfEMP1) family and receptors on the endothelial membrane. CM is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13. However, the major endothelial receptor for parasites exhibiting these ligands remains controversial but may include endothelial protein C receptor (EPCR) and intercellular adhesion molecule 1 (ICAM1). Previous work suggests complement C1q binding protein (C1QBP) as a potential binding partner for IEs, but this has rarely been studied. In this thesis, I used a) HBECs and immunofluorescence assays to determine the cellular localisation of C1QBP and b) adhesion assays to determine the role of C1QBP in IE (IT4VAR19, HB3VAR03, 9197VAR27, and PfKE08) adhesion to hCMEC/D3. Firstly, I validated the endothelial nature of hCMEC/D3 with specific endothelial cell markers such as von Willebrand factor (vWF), PECAM1/CD31 (Platelet endothelial cell adhesion molecule 1), Dil-Ac-LDL (Dil-Acetylated- low density lipoprotein), and SMA (Smooth muscle actin). As expected hCMEC/D3 exhibited positive staining for vWF, PECAM1/CD31, Dil-Ac-LDL, and a negative staining for SMA. Therefore, I can use hCMEC/D3 as an endothelial cell to determine the cellular localisation of C1QBP. Resting and TNFα-activated hCMEC/D3 showed intracellular staining for C1QBP, but cell surface staining was not observed. However, after incubation with soluble C1QBP or normal human plasma (which contains soluble C1QBP), hCMEC/D3 did exhibit positive surface membrane expression of C1QBP. In addition, I observed that primary HBECs constitutively express C1QBP but not immortalised HBECs. These results show that primary HBECs may exhibit different surface receptors compared to immortalised HBECs. Secondly, I investigated whether C1QBP can promote IE binding to hCMEC/D3. In static binding assays to purified receptors, IT4VAR19-IE showed significant binding to C1QBP, TSP (thrombospondin) and EPCR (endothelial protein C receptor). In adhesion inhibition assays, I found that a monoclonal antibody (mAb) to human EPCR blocked the interaction between III IT4VAR19-IE and hCMEC/D3, whereas a mAb and polyclonal (pAb) antibody to human C1QBP did not. HB3VAR03-IE did not interact with C1QBP and other potential endothelial cell receptors, while 9197VAR27 and PFKE08-IE did not also bind to C1QBP but significantly adhered to EPCR. These data do not support a role for C1QBP as a major endothelial cell cytoadhesion receptor for the four IEs lines studied here. In conclusion, C1QBP is not constitutively expressed on the surface of immortalised HBECs but can become associated with the cell surface membrane after exposure to normal human plasma or recombinant C1QBP. C1QBP is not the major host receptor for IEs tested here but may act in combination with EPCR. Since primary HBEC constitutively expressed C1QBP, IE may have developed the capacity to interact with C1QBP to prevent the activation of the classical pathway of the complement system and thus ensures its survival and transmission.

https://era.ed.ac.uk/handle/1842/44528

https://doi.org/10.7488/era/7045

en

McLean, F. E., Omondi, B. R., Diallo, N., Otoboh, S., Kifude, C., Abdi, A. I., Lim, R., Otto, T. D., Ghumra, A., & Rowe, J. A. (2025). Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype. PLoS Pathog, 21(1), e1012434. https://doi.org/10.1371/journal.ppat.1012434

C1QBP

Plasmodium falciparum

HBECs (Human Brain Microvascular Endothelial Cells)

Adhesion

EPCR (Endothelial Protein C Receptor)

Role of C1QBP for Plasmodium falciparum infected erythrocyte binding to human brain microvascular endothelial cells

Investigating the role of C1QBP for Plasmodium falciparum infected erythrocyte binding to human brain microvascular endothelial cells

Thesis

Masters

MPhil Master of Philosophy