Background:
Plasmodium falciparum
, the etiologic agent of malaria, is a major cause of infant death in Africa. Although research on the contact system has been revitalized by recent discoveries in the field of thrombosis, limited efforts were done to investigate the role of its proinflammatory arm, the kallikrein kinin system (KKS), in the pathogenesis of neglected parasitic diseases, such as malaria. Owing to the lack of animal models, the dynamics of central nervous system (CNS) pathology caused by the sequestration of erythrocytic stages of
P. falciparum
is not fully understood. Given the precedent that kinins destabilize the blood brain barrier (BBB) in ischemic stroke, here we sought to determine whether
Plasmodium falciparum
infected erythrocytes (
Pf
-iRBC) conditioned medium enhances parasite sequestration and impairs BBB integrity via activation of the kallikrein kinin system (KKS).
Methods:
Monolayers of human brain endothelial cell line (BMECs) are preincubated with the conditioned medium from
Pf
-iRBCs or RBCs (controls) in the presence or absence of HOE-140 or DALBK, antagonists of bradykinin receptor B2 (B2R) and bradykinin receptor B1 (B1R), respectively. Following washing, the treated monolayers are incubated with erythrocytes, infected or not with
P. falciparum
mature forms, to examine whether the above treatment (i) has impact on the adhesion of
Pf
-iRBC to BMEC monolayer, (ii) increases the macromolecular permeability of the tracer BSA-FITC, and (iii) modifies the staining pattern of junctional proteins (ZO-1 and β-catenin).
Results:
We found that kinins generated in the parasite conditioned medium, acting via bradykinin B2 and/or B1 receptors (i) enhanced
Pf
-iRBC adhesion to the endothelium monolayer and (ii) impaired the endothelial junctions formed by ZO-1 and β-catenin, consequently disrupting the integrity of the BBB.
Conclusions:
Our studies raise the possibility that therapeutic targeting of kinin forming enzymes and/or endothelial bradykinin receptors might reduce extent of
Pf
-iRBC sequestration and help to preserve BBB integrity in cerebral malaria (CM).
To
cope up with toxic effects of heme generation during hemoglobin
catabolism, malaria parasites immobilize heme molecules in an inert
form known as hemozoin crystal. This mechanism is essential for parasite
development and represents a physiological step used as a target for
many antimalarial drugs. So far, most of the data on hemozoin structure
and elemental composition has been obtained through the analysis of
β-hematin, a synthetic analogue of hemozoin whose structure
and physical properties have been the subject of many studies. Fundamental
questions regarding crystal growth, immunomodulatory effects, and
drug inhibition mechanisms, nevertheless, remain unanswered, especially
considering crystals obtained from different malaria species. In this
work, we used high-resolution electron microscopy approaches to analyze
hemozoin crystals isolated from malaria murine models and compared
their structure to isolated crystals obtained from the human malaria
parasite Plasmodium falciparum. A comparative
analysis of the elemental composition of Hz crystals (isolated or
in situ) in murine models was also carried out. Results showed that
hemozoin crystals in different Plasmodium species
presented a rectangular morphology, with significant size differences
between murine and human malaria species. A detailed structural and
chemical analysis of the surface of the crystals showed growing areas,
indicating points where antimalarial drugs may interfere. Elemental
analysis showed that crystals obtained from different species are
chemically similar, suggesting that they may potentially perform similar
immunomodulatory functions.
1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 μM. The inhibitory effect of 972 μM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.
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