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Page 6 of 15 Cheng et al. Vessel Plus 22020;4:17 I http://dx.doi.org/10.20517/2574-1209.2020.08
[76]
syndrome in C57BL/6 mice . These animals displayed basal levels of plasma MVs but numbers failed
to increase following infection (as observed in wild type counterparts), had lower levels of plasma TNF,
reduced expression of endothelial cell adhesion molecules and had increased survival of leukocytes and
platelets. Another study examining the blocking of phosphatidylserine using low-molecular-weight thiol
pantethine found a similar reduction in MV production, which correlated with reduction in inflammation
[78]
and resistance to the disease . When passively transferred into the circulation of mice, plasma MVs from
infected animals localised to the inflamed vessels of infected animals, notably in the brain, which suggests
that they could potentiate the neurovascular lesions by interacting with other vascular cells. In addition,
healthy mice injected with TNF-generated endothelial MVs developed CM-like pathology with cerebral
and pulmonary oedema and haemorrhage, the two main histopathological features of human and murine
CM [77,79,80] .

Interaction with and internalisation of MVs derived from iRBCs by astrocytes and microglial cells leads
to increased production of IFN-γ-inducible protein 10 (IP-10), which coincided with increased levels of
[81]
inflammatory cytokines within both plasma and brain tissue of PbA-infected mice . Plasma MVs from
PbA-infected mice were also able to activate immune cells, in particular macrophages, leading to the up-
[54]
regulation of CD40 as well as TNF production . Knock-out mice that lack pro-inflammatory cytokines
-/-
(TNF , IFN-γ , IL-12 and RAG-1 ) displayed levels of plasma MVs similar to those of their wild-type
-/-
-/-
-/-
[54]
counterparts , suggesting that their production is not solely dependent on the presence of inflammation.
When exosomes, purified from the blood of P. yoelii (nonlethal strain,17XNL)-infected mice, were injected
into mice infected with P. yoelii (lethal strain, 17XL), these mice were protected against the lethal syndrome,
showing that exosomes could also modulate the immune response [82,40] . We propose a model in Figure 1
that summaries the role EVs play in the pathogenesis of CM.

Figure 1 was designed by modifying free images provided by Smart Servier Medical Art (https://smart.
servier.com/) and available under the creative commons license.

EXTRACELLULAR VESICLE CARGO: EFFECTOR, BIOMARKER OR BOTH?
Biomarkers have long been used as diagnostic and prognostic tools to determine the presence of disease
[85]
as well as the regression, progression or outcome after treatment . The field of malaria, especially
severe malaria, currently lacks reliable markers for the prediction of morbidities such as neurocognitive
impairment and/or mortality that can be widely applicable regardless of country or endemicity. Such
biomarkers would allow for the prediction of severe complications and allow early implementation
of adjunctive therapies. Current adjunctive therapies used to aid anti-malarial drugs have so far been
[86]
ineffective , possibly due to late implementation due to the lack of predictive biomarkers. Therefore, early
identification of patients at risk of severe malaria complications (lethal or not) would allow for prompt
treatment and potentially decrease the risk of long-term disabilities.

Currently, only a handful of candidate markers have been identified for severe malaria, though they are
not fully reliable. The proposed molecular markers include erythropoietin, angiopoietin 2, von Willebrand
factor, P. falciparum histidine-rich protein 2 and ICAM-1 which, although indicative of severe disease when
elevated, still has limited clinical utility. This is mainly due to the highly variable sensitivity and specificity
for the detection of severe CM [41,87-90] . It is now clear that EVs and their cargo have potential as biomarkers.
Their elevated numbers, notably endothelial-, platelet- and RBC-derived MVs, in the circulation of human
patients with CM has already been proven in multiple studies [39,84,91] . The role of EVs as a biomarker in
severe malaria is still in its infancy and in-depth multi-centre studies are still needed to ascertain their
predictive value to improve rapid detection in bodily fluids. In addition, although blood, urine, and saliva
have all been used for diagnostics [92,93] , urine and saliva have not been investigated in malaria but we