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Platelets Reorient Plasmodium falciparum-Infected Erythrocyte Cytoadhesion to Activated Endothelial Cells
Abstract
Severe malaria is characterized by the sequestration of Plasmodium falciparum–infected erythrocytes (IEs). Because platelets can affect tumor necrosis factor (TNF)–activated endothelial cells (ECs),
we investigated their role in the sequestration of IEs, using IEs that were selected because they can adhere to endothelial
CD36 (IECD36), a P. falciparum receptor that is expressed on platelets. The results of coincubation studies indicated that platelets can induce IECD36 binding to CD36-deficient brain microvascular ECs. This induced cytoadhesion resisted physiological shear stress, was increased
by EC stimulation with TNF, and was abolished by anti–CD36 monoclonal antibody. Immunofluorescence and scanning electron microscopy
results showed that platelets serve as a bridge between IEs and the surface of ECs and may therefore provide receptors for
adhesion to microvascular beds that otherwise lack adhesion receptors. This novel mechanism of cytoadhesion may reorient the
sequestration of different parasite phenotypes and play an important role in the pathogenesis of severe malaria
... Furthermore, autoagglutinates formed by CM isolates were much bigger than those formed by isolates from patients without CM, suggesting that they may cause microcirculatory obstruction and impair blood flow in the brain. Platelets may also act as bridges between iRBCs and ECs and, as such, mediate endothelial cytoadhesion in the microvasculature of organs devoid of specific receptors such as the brain that normally does not express CD36 (Wassmer et al. 2004). Autoagglutination has also been found in blood samples of P. vivax patients, but the mechanism of this has not been described yet (Jayavanth and Park 2007). ...
... However, P. falciparum and P. vivax patients with severe malaria infections have significantly reduced ADAMTS13-activity (de Mast et al. 2009;Larkin et al. 2009;Lowenberg et al. 2010;Barber et al. 2015). Thus, the combination of endothelial activation with increased circulating amounts of active and UL-vWF, together with reduced vWF inactivation by ADAMTS13, may result in intravascular platelet aggregation, as observed in brains and lungs of severely ill patients and mice (Grau et al. 1993(Grau et al. , 2003Sun et al. 2003;Wassmer et al. 2004;von Zur Muhlen et al. 2008;Hochman et al. 2015) and in P. falciparum-infected placentas (Wassmer et al. 2004). In P. vivax patients, platelet numbers are also positively correlated with ANG1 and negatively with ANG2 and the ANG2/ANG1 ratio . ...
... However, P. falciparum and P. vivax patients with severe malaria infections have significantly reduced ADAMTS13-activity (de Mast et al. 2009;Larkin et al. 2009;Lowenberg et al. 2010;Barber et al. 2015). Thus, the combination of endothelial activation with increased circulating amounts of active and UL-vWF, together with reduced vWF inactivation by ADAMTS13, may result in intravascular platelet aggregation, as observed in brains and lungs of severely ill patients and mice (Grau et al. 1993(Grau et al. , 2003Sun et al. 2003;Wassmer et al. 2004;von Zur Muhlen et al. 2008;Hochman et al. 2015) and in P. falciparum-infected placentas (Wassmer et al. 2004). In P. vivax patients, platelet numbers are also positively correlated with ANG1 and negatively with ANG2 and the ANG2/ANG1 ratio . ...
Coevolution of humans and malaria parasites has generated an intricate balance between the immune system of the host and virulence factors of the parasite, equilibrating maximal parasite transmission with limited host damage. Focusing on the blood stage of the disease, we discuss how the balance between anti-parasite immunity versus immunomodulatory and evasion mechanisms of the parasite may result in parasite clearance or chronic infection without major symptoms, whereas imbalances characterized by excessive parasite growth, exaggerated immune reactions or a combination of both cause severe pathology and death, which is detrimental for both parasite and host. A thorough understanding of the immunological balance of malaria and its relation to other physiological balances in the body is of crucial importance for developing effective interventions to reduce malaria-related morbidity and to diminish fatal outcomes due to severe complications. Therefore, we discuss in this review the detailed mechanisms of anti-malarial immunity, parasite virulence factors including immune evasion mechanisms and pathogenesis. Furthermore, we propose a comprehensive classification of malaria complications according to the different types of imbalances.
... Infected erythrocytes can bind to platelets and the platelets function as bridges between infected erythrocytes [143]. Platelets also bind to endothelial cells and can function as bridges between infected erythrocytes and endothelial cells [144]. Three potential PfEMP1 receptors for platelet mediated clumping are CD36 [143,144], gC1qR/HABP1/p32 [114], and P-selectin [145]. ...
... Platelets also bind to endothelial cells and can function as bridges between infected erythrocytes and endothelial cells [144]. Three potential PfEMP1 receptors for platelet mediated clumping are CD36 [143,144], gC1qR/HABP1/p32 [114], and P-selectin [145]. Other potential receptors that are associated with platelets include platelet endothelial cell adhesion molecule-1, E-selectin, and thrombospondin. ...
Plasmodium falciparum can cause a severe disease with high mortality. A major factor contributing to the increased virulence of P. falciparum, as compared to other human malarial parasites, is the sequestration of infected erythrocytes in the capillary beds of organs and tissues. This sequestration is due to the cytoadherence of infected erythrocytes to endothelial cells. Cytoadherence is primarily mediated by a parasite protein expressed on the surface of the infected erythrocyte called P. falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 is embedded in electron-dense protuberances on the surface of the infected erythrocytes called knobs. These knobs are assembled on the erythrocyte membrane via exported parasite proteins, and the knobs function as focal points for the cytoadherence of infected erythrocytes to endothelial cells. PfEMP1 is a member of the var gene family, and there are approximately 60 antigenically distinct PfEMP1 alleles per parasite genome. Var gene expression exhibits allelic exclusion, with only a single allele being expressed by an individual parasite. This results in sequential waves of antigenically distinct infected erythrocytes and this antigenic variation allows the parasite to establish long-term chronic infections. A wide range of endothelial cell receptors can bind to the various PfEMP1 alleles, and thus, antigenic variation also results in a change in the cytoadherence phenotype. The cytoadherence phenotype may result in infected erythrocytes sequestering in different tissues and this difference in sequestration may explain the wide range of possible clinical manifestations associated with severe falciparum malaria.
... The activated endothelial cells release cytokines that upregulate the expression of adhesion molecules (P-, L-and E-selectin) (Combes et al., 2004;Krishnamurthy et al., 2015). Chemokines such as CCL2, CXCL4, CCL5 (Van den Steen et al., 2010), promote the adhesion of iRBCs, macrophages (Grau et al., 1987), platelets (Wassmer et al., 2004), neutrophils (Souza et al., 2013) and plasma microparticles (Faille et al., 2009) to the endothelium. IFN-g, TNF-a and IL-1, produced by NK and mononuclear cells (alveolar and interstitial macrophages), are the most prevalent cytokines involved in leukocytes chemotaxis and for ICAM-1/ CD54 and VCAM-1/CD106 expression on the endothelial cells (Smith et al., 2019). ...
... The interaction between the platelet and iRBCs is mediated through CD31/PECAM-1 and CD36 expressed on platelets with PfEMP-1 expressed on iRBCs (Pain et al., 2001). Platelets also can act as an adhesive bridge between P. falciparum-iRBCs and activated endothelial cells (Wassmer et al., 2004). Studies done either in vitro using human platelets or in Plasmodium-infected mice depleted of platelets demonstrated that platelets are responsible for the killing of iRBCs, a mechanism involving platelet factor 4 (PF4/CXCL4) (McMorran et al., 2009;McMorran et al., 2012). ...
Malaria-associated acute respiratory distress syndrome (MA-ARDS) is increasingly gaining recognition as a severe malaria complication because of poor prognostic outcomes, high lethality rate, and limited therapeutic interventions. Unfortunately, invasive clinical studies are challenging to conduct and yields insufficient mechanistic insights. These limitations have led to the development of suitable MA-ARDS experimental mouse models. In patients and mice, MA-ARDS is characterized by edematous lung, along with marked infiltration of inflammatory cells and damage of the alveolar-capillary barriers. Although, the pathogenic pathways have yet to be fully understood, the use of different experimental mouse models is fundamental in the identification of mediators of pulmonary vascular damage. In this review, we discuss the current knowledge on endothelial activation, leukocyte recruitment, leukocyte induced-endothelial dysfunction, and other important findings, to better understand the pathogenesis pathways leading to endothelial pulmonary barrier lesions and increased vascular permeability. We also discuss how the advances in imaging techniques can contribute to a better understanding of the lung lesions induced during MA-ARDS, and how it could aid to monitor MA-ARDS severity.
... 16,24 Second, de Mast et al have demonstrated that in patients with P falciparum infection, a significant proportion of plasma VWF is circulating in an active confirmation that promotes platelet glycoprotein Ib binding. 24 This observation is important because platelet adhesion and accumulation have been implicated in facilitating the adhesion of P falciparum IE to activated EC. [25][26][27] Finally, in a shear-based assay, we have recently shown that platelet-decorated UL-VWF strings on the surface of activated EC can also recruit trophozoitestage P falciparum IE. 28 The early increase in plasma VWF levels and circulating UL-VWF multimers observed following P falciparum infection poses a challenge in defining whether VWF directly contributes to the development of human CM or whether increased VWF levels merely constitute a secondary epiphenomenon associated with EC activation. In this study, we have sought to further investigate the putative role of VWF in malaria pathogenesis in vivo using an established murine model of experimental cerebral malaria (ECM). ...
... 72 In addition, plasma ADAMTS13 activity levels are reduced in some strains (including C57BL/6) because the insertion of a retrotransposon element into the ADAMTS13 gene. 73,74 Given the key role played by VWF in modulating platelet adhesion and aggregation, together with the accumulating data demonstrating that platelet-EC interactions play a critical role in malaria biology, [25][26][27] we hypothesized that VWF may play a novel role in modulating the pathophysiology of malaria. This hypothesis was supported by previous in vitro experiments in which we showed that plateletdecorated UL-VWF strings could recruit P falciparum IE to EC surfaces under shear stress conditions. ...
Key Points
ECM is associated with an early marked increase in plasma VWF levels and accumulation of UL-VWF multimers. Following P berghei infection, VWF−/− mice survive significantly longer compared with WT controls.
... Furthermore, platelets play an important role in the induction of the clumping of iRBCs [54] and in vitro induce the cytoadherence of iRBCs to endothelial cells [55,56]. Interestingly, platelet-EVs bind to iRBCs and transfer platelet antigens into the infected cells; binding occurs only to iRBCs in a PfEMP1 dependent-manner, whereas platelet-EVs do not bind to uninfected RBCs. ...
... IFN-γ may mediate the development of experimental cerebral malaria through various mechanisms. IFN-γ, together with tumor necrosis factor (TNF) and lymphotoxin α, enhance the activation and apoptosis of the brain endothelium through the activation of endothelial cell and subsequently increased local binding of platelets [64,127,128]. IFN-γ is also necessary for the recruitment of CD8+ T cells in the brain by inducing the expression of canonical adhesion molecules, such as ICAM-1, CXCL9, and CXCL10. Accumulated CD8+ T cells mediate the immune responses against infected red blood cells sequestered in the brain and the lungs in susceptible mice, facilitating development of experimental CM [61,63]. ...
Protozoan parasites, such as Plasmodium, Leishmania, Toxoplasma, Cryptosporidium, and Trypanosoma, are causative agents of health-threatening diseases in both humans and animals, leading to significant health risks and socioeconomic losses globally. The development of effective therapeutic and prevention strategies for protozoan-caused diseases requires a full understanding of the pathogenesis and protective events occurring in infected hosts. Interferons (IFNs) are a family of cytokines with diverse biological effects in host antimicrobial defense and disease pathogenesis, including protozoan parasite infection. Type II IFN (IFN-γ) has been widely recognized as the essential defense cytokine in intracellular protozoan parasite infection, whereas recent studies also revealed the production and distinct function of type I and III IFNs in host defense against these parasites. Decoding the complex network of the IFN family in host–parasite interaction is critical for exploring potential new therapeutic strategies against intracellular protozoan parasite infection. Here, we review the complex effects of IFNs on the host defense against intracellular protozoan parasites and the crosstalk between distinct types of IFN signaling during infections.
... The magnitude of the recovery was greater than expected by a passive transfer of platelets contained in the transfused blood. Because platelets have been implicated with the pathogenesis of ECM, in processes such as coagulopathy, (49) endothelial activation, (34) cytoadherence (50) and auto-agglutination, (51) restoring fresh platelets in a timely manner may be helpful in preventing mortality. ...
Background:
Cerebral malaria is a lethal complication of Plasmodium falciparum infections in need of better therapies. Previous work in murine experimental cerebral malaria (ECM) indicated that the combination of artemether plus intraperitoneal whole blood improved vascular integrity and increased survival compared to artemether alone. However, the effects of blood or plasma transfusion administered via the intravenous route have not previously been evaluated in ECM.
Objectives:
To evaluate the effects of intravenous whole blood compared to intravenous plasma on hematological parameters, vascular integrity, and survival in artemether-treated ECM.
Methods:
Mice with late-stage ECM received artemether alone or in combination with whole blood or plasma administered via the jugular vein. The outcome measures were hematocrit and platelets; plasma angiopoietin 1, angiopoietin 2, and haptoglobin; blood-brain barrier permeability; and survival.
Findings:
Survival increased from 54% with artemether alone to 90% with the combination of artemether and intravenous whole blood. Intravenous plasma lowered survival to 18%. Intravenous transfusion provided fast and pronounced recoveries of hematocrit, platelets, angiopoietins levels and blood brain barrier integrity.
Main conclusions:
The outcome of artemether-treated ECM was improved by intravenous whole blood but worsened by intravenous plasma. Compared to prior studies of transfusion via the intraperitoneal route, intravenous administration was more efficacious.
... P-selectin increases the adherence of infected RBCs to CD36 on the endothelium [44,45] and may act as a trigger for the amplification of the clumping phenomenon. Platelets can form bridges between endothelial cells and infected RBCs [32,46,47] after bonding to the TNF activated endothelial cells with the help of specific molecules such as endothelial CD40 and platelet CD41. In addition to direct cytoadherence (infected RBCs to the endothelial wall), platelet-associated clumping and bridging may also contribute to the obstruction of the brain microvessels in CM patients. ...
The severe form of malaria, i.e., cerebral malaria caused by Plasmodium falciparum, is a complex neurological syndrome. Surviving persons have a risk of behavioral difficulties, cognitive disorders, and epilepsy. Cerebral malaria is associated with multiple organ dysfunctions. The adhesion and accumulation of infected RBCs, platelets, and leucocytes (macrophages, CD4+ and CD8+ T cells, and monocytes) in the brain microvessels play an essential role in disease progression. Micro-vascular hindrance by coagulation and endothelial dysfunction contributes to neurological damage and the severity of the disease. Recent studies in human cerebral malaria and the murine model of cerebral malaria indicate that different pathogens as well as host-derived factors are involved in brain microvessel adhesion and coagulation that induces changes in vascular permeability and impairment of the blood-brain barrier. Efforts to alleviate blood-brain barrier dysfunction and de-sequestering of RBCs could serve as adjunct therapies. In this review, we briefly summarize the current understanding of the pathogenesis of cerebral malaria, the role of some factors (NK cells, platelet, ANG-2/ANG-1 ratio, and PfEMP1) in disease progression and various functions of Mesenchymal stem cells. This review also highlighted the implications of MSCs as a regenerative medicine.
... Most in vitro models of CM simulate the interactions between microvascular endothelial cells and circulating vascular cells (e.g., iRBCs, nRBCs, platelets, and leucocytes) in either static or shear stress environments [41,21] . The brain endothelial cells used can be of human, simian or murine origin (primary or immortalised), and co-cultured with one or more other cell types in two-dimensional systems [42][43][44][45] . The recent introduction of more complex three-dimensional models will help to examine and understand the pathogenesis of this disease better [46][47][48] . ...
Malaria infections due to the Plasmodium parasite remains a major global health problem. Plasmodium falciparum is responsible for majority of the severe cases, resulting in more than 400,000 deaths per annum. Extracellular vesicles (EVs) released by vascular cells, including parasitised erythrocytes, have been detected with increased levels in patients with malaria. EVs are thought to be involved in the pathogenesis of severe malaria, particularly cerebral malaria, and represent a unique molecular signature for different forms of the infection. In this review, we will cover the known effects of EVs on the vasculature and discuss their potential use as a biomarker of disease severity.
... But, again, the effect of intravenous blood transfusion was stronger, leading to a faster recovery of platelet counts and again the magnitude of the recovery was greater than it would be expected by a passive transfer of platelets contained in the transfused blood. Because platelets have been implicated with the pathogenesis of ECM, in processes such as coagulopathy [48], endothelial activation [32], cytoadherence [49] and auto-agglutination [50], restoring fresh platelets in a timely manner may be helpful in preventing mortality. under 17 USC 105 and is also made available for use under a CC0 license. ...
Transfusion of 10 mg/kg of whole blood via intraperitoneal route to mice with late-stage experimental cerebral malaria (ECM) along with artemether has been shown to result in markedly increased survival (75%) compared to artemether alone (51%). Intraperitoneal route was used to overcome the restrictions imposed by injection of large volumes of viscous fluid in small and deranged blood vessels of mice with ECM. In the present study, a method of intravenous transfusion was implemented by injecting 200mL of whole blood through the right jugular vein in mice with late-stage ECM, together with artemether given intraperitoneally, leading to a remarkable increase in survival, from 54% to 90%. On the contrary, mice receiving artemether plus plasma transfusion showed a worse outcome, with only 18% survival. Compared to the intraperitoneal route, intravascular transfusion led to faster and more pronounced recoveries of hematocrit, platelet counts, angiopoietins levels (ANG-1, ANG-2 and ANG-2/ANG-1) and blood brain barrier integrity. These findings indicate that whole blood transfusion when given intravenously show more efficacy over intraperitoneal transfusion, reinforcing evidence for benefit as an adjuvant therapy for cerebral malaria.
... In experimental and human CM, studies have shown that platelets account for more than 50 % of total plasma EVs [33,38,89]. Moreover, since platelets can bridge interactions between endothelial cells and iRBCs by providing the necessary surface adhesion molecules [90] that are also present and enriched on the surface of PEVs [18,72], it was postulated that elevated circulating PEVs was an indication of their potential role in the pathogenesis of CM. This postulation was eloquently investigated in an in vitro model of HCM by Faille and colleagues [91]. ...
Malaria is a complex parasitic disease, caused by Plasmodium spp. More than a century after the discovery of malaria parasites, this disease continues to pose a global public health problem and the pathogenesis of the severe forms of malaria remains incompletely understood. Extracellular vesicles (EVs), including exosomes and microvesicles, have been increasingly researched in the field of malaria in a bid to fill these knowledge gaps. EVs released from Plasmodium -infected red blood cells and other host cells during malaria infection are now believed to play key roles in disease pathogenesis and are suggested as vital components of the biology of Plasmodium spp. Malaria-derived EVs have been identified as potential disease biomarkers and therapeutic tools. In this review, key findings of malaria EV studies over the last 20 years are summarized and critically analysed. Outstanding areas of research into EV biology are identified. Unexplored EV research foci for the future that will contribute to consolidating the potential for EVs as agents in malaria prevention and control are proposed.
... Remarkably, platelet-derived exosomes are able to alter their cargo contents during inflammation, for example, they increase levels of the agranule chemokines CXCL4 and CXCL7 (25)(26)(27). This may be the mechanism that enables exosomes to induce blood cells clumping in responses to malaria (28,29). Ongoing technological and experimental advances are likely to yield more discoveries regarding exosome functional heterogeneity. ...
Schistosomiasis is a parasitic disease endemic to freshwater areas of Southeast Asia, Africa, and South America that is capable of causing serious damage to the internal organs. Recent studies have linked exosomes to the progression of schistosomiasis. These structures are important mediators for intercellular communication, assist cells to exchange proteins, lipids, and genetic material and have been shown to play critical roles during host–parasite interactions. This review aims to discuss the pathophysiology of exosomes in schistosomiasis and their roles in regulating the host immune response. Understanding how exosomes are involved in the pathogenesis of schistosomiasis may provide new perspectives in diagnosing and treating this neglected disease.
... They also release microparticles that potentially inhibit parasite growth [49]. Platelets can bridge the binding of iRBCs without tropism to the brain endothelium [50] and may promote microvascular obstruction by platelet-mediated clumping of iRBCs [51] or iRBC binding to platelet-von Willebrand Factor (vWF) multimers secreted upon endothelial activation or injury [52] (Figure 2). ...
Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication that accounts for most of the malaria deaths worldwide. P. falciparum sequestration in the brain microvasculature is a hallmark of CM and is not replicated in animal models. Numerous aspects of the disease are challenging to fully understand from clinical studies, such as parasite binding tropism or causal pathways in blood–brain barrier breakdown. Recent bioengineering approaches allow for the generation of 3D microvessels and organ-specific vasculature that provide precise control of vessel architecture and flow dynamics, and hold great promise for malaria research. Here, we discuss recent and future applications of bioengineered microvessels in malaria pathogenesis research.
... The cysteine-rich interdomain region (CIDR) and Duffy binding-like (DBL) domains (Mkumbaye et al., 2017;Shabani et al., 2017;Tuikue Ndam et al., 2017) are located at the N-terminal of the extracellular portion of PfEMP1. The binding of PfEMP1 to different host receptors determines the amount of sequestration and is associated with disease severity (Newbold et al., 1997;Wassmer et al., 2004;Ochola et al., 2011). PfEMP1 has been found to bind to CD36 on platelets via CIDRa2-6 domains in uncomplicated/mild malaria (UM/MM) (Mkumbaye et al., 2017;Mustaffa et al., 2017), and to ICAM-1 on endothelial cells in severe malaria (SM) via DBLb5 domains (Mustaffa et al., 2017;Tuikue Ndam et al., 2017). ...
Background
Cerebral malaria (CM) is characterized by the sequestration of Plasmodium-infected erythrocytes (pRBCs) to host brain microvasculature beds via P. falciparum erythrocyte membrane protein 1 (PfEMP1). Under normal conditions, activated protein C (APC) bound to endothelial protein C receptor (EPCR) has cytoprotective properties via the activation of protease-activated receptor 1 (PAR1). During malaria infection, pRBCs transports PfEMP1 to the membranes to bind EPCR in the same region as APC. As a result, APC is less capable of inducing cytoprotective effects via PAR1. Two studies involving adult malaria patients revealed that EPCR rs867186-GG allele is associated with protection against severe malaria, while three other studies involving child malaria patients could not show association between EPCR rs867186-GG genotype and severe malaria or increased mortality among children with CM.
Methods
We examined the association between the EPCR rs867186-GG genotype and the protection against cerebral malaria. Peripheral blood samples were collected from 47 malaria patients and 34 healthy individuals from a study conducted from 2004 to 2007 at the NSCB Medical College Hospital in India. CM and malaria-associated complications were defined based on WHO criteria. Genomic DNA was isolated from the peripheral blood mononuclear cells. Primer sequences were designed to contain rs867186 of the PROCR gene (NM 006404) and were used to amplify a 660 bp product as described before. PCR products were purified, and DNA sequences were determined by Sanger Sequencing (Genewiz, NJ). Nonparametric tests were used to compare the groups. To analyze differences in allele frequencies, we used chi-squared or Fisher's exact tests for categorical variables if the expected values were less than 5. P-value <0.05 was considered statistically significant.
Results
Our results showed significantly higher rates of AG and GG genotypes in CM patients compared to mild malaria (P = 0.0034).
Conclusion
Our results indicate that rs867186-GG or rs867186-AG genotypes are not associated with protection against HCM.
... Interestingly, VWF strings have been proposed to facilitate parasite sequestration, which can induce further inflammation and endothelial activation [15]. VWF-adhering platelets can for example bind to infected red blood cells (iRBCs) and bridge iRBCs to the endothelium, which may aid the parasite to evade splenic clearance [16,17]. ...
Background:
Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an understudied complication of malaria and is characterized by pulmonary inflammation and disruption of the alveolar-capillary membrane. Its pathogenesis remains poorly understood. Since endothelial activation plays an important role in other malarial complications, the expression of two endothelial activation markers, von Willebrand factor (VWF) and angiopoietin-2 (ANG-2), was investigated in the lungs of patients with MA-ARDS.
Methods:
Post-mortem lung sections of Plasmodium falciparum-infected patients without alveolar oedema (NA), P. falciparum-infected patients with alveolar oedema (MA-ARDS), and uninfected people who died accidentally with no pathological changes to the lungs (CON) were immunohistochemically stained for VWF and ANG-2, and were evaluated with semi-quantitative analysis.
Results:
Alveolar oedematous VWF and ANG-2 and intravascular VWF staining were significantly increased in patients with MA-ARDS versus infected and uninfected control groups. The levels of VWF in the alveolar septa and endothelial lining of large blood vessels of patients with MA-ARDS was significantly decreased compared to controls. ANG-2 expression was increased in the alveolar septa of malaria patients without alveolar oedema versus control patients, while ANG-2+ leukocytes were increased in the alveoli in both infected patient groups.
Conclusions:
This study documents a high level of VWF and ANG-2, two endothelial activation markers in the oedematous alveoli of post-mortem lung sections of Thai patients with MA-ARDS. Decreased detection of VWF in the endothelial lining of blood vessels, in parallel with an increased presence of intravascular VWF staining suggests marked endothelial activation and Weibel-Palade body release in the lungs of patients with MA-ARDS.
... Even though endothelial CD36 is present in very low amounts in cerebral malaria patients (8), IRBC-CD36 binding affinity was 10 times that of IRBC-ICAM-1 bonds at room temperature (13). With our current results that show nonsubstantial differences in binding affinities at physiologically relevant temperatures, it seems much less likely that CD36 plays any significant role in cerebral sequestration, be it expressed on endothelium or platelets (56,57). The contrasting conclusions derived from data collected at different temperatures further emphasizes the importance of examining IRBC-receptor interactions at physiologically relevant temperatures. ...
Plasmodium falciparum malaria-infected red blood cells (IRBCs), or erythrocytes, avoid splenic clearance by adhering to host endothelium. Upregulation of endothelial receptors intercellular adhesion molecule-1 (ICAM-1) and cluster of differentiation 36 (CD36) are associated with severe disease pathology. Most in vitro studies of IRBCs interacting with these molecules were conducted at room temperature. However, as IRBCs are exposed to temperature variations between 37°C (body temperature) and 41°C (febrile temperature) in the host, it is important to understand IRBC-receptor interactions at these physiologically relevant temperatures. Here, we probe IRBC interactions against ICAM-1 and CD36 at 37 and 41°C. Single bond force-clamp spectroscopy is used to determine the bond dissociation rates and hence, unravel the nature of the IRBC-receptor interaction. The association rates are also extracted from a multiple bond flow assay using a cellular stochastic model. Surprisingly, IRBC-ICAM-1 bond transits from a catch-slip bond at 37°C toward a slip bond at 41°C. Moreover, binding affinities of both IRBC-ICAM-1 and IRBC-CD36 decrease as the temperature rises from 37 to 41°C. This study highlights the significance of examining receptor-ligand interactions at physiologically relevant temperatures and reveals biophysical insight into the temperature dependence of P. falciparum malaria cytoadherent bonds.
... The IRBC-cytoadherence events are usually classified based on their binding sites, i.e., endothelial cytoadhesion, cytoadhesion to placental syncytiotrophoblasts, platelets, URBCs (rosetting phenomenon) and leukocytes (monocytes, macrophages, and dendritic cells) (23, [60][61][62]. P. falciparum IRBCs can adhere to each other through platelet bridges, forming aggregates of IRBCs, a mechanism defined as autoagglutination (63)(64)(65). This phenomenon has been shown to be uncorrelated to rosetting and parasitemia, but significantly associated with severe malaria (63). ...
After a successful invasion, malaria parasite Plasmodium falciparum extensively remodels the infected erythrocyte cellular architecture, conferring cytoadhesive properties to the infected erythrocytes. Cytoadherence plays a central role in the parasite's immune-escape mechanism, at the same time contributing to the pathogenesis of severe falciparum malaria. In this review, we discuss the cytoadhesive interactions between P. falciparum infected erythrocytes and various host cell types, and how these events are linked to malaria pathogenesis. We also highlight the limitations faced by studies attempting to correlate diversity in parasite ligands and host receptors with the development of severe malaria.
... In the same way, platelet adhesion to brain endothelial cells has been associated with cerebral malaria (Grau et al., 2003). Furthermore, it has been shown to promote adhesion of infected red blood cells to TNF-stimulated endothelium (Wassmer et al., 2004), and to alter gene expression in vitro in human brain microvascular endothelial cells (Barbier et al., 2011), thus contributing to brain endothelial dysfunction. ...
Background
Host factors, including host genetic variation, have been shown to influence the outcome of Plasmodium falciparum infection. Genome-wide linkage studies have mapped mild malaria resistance genes on chromosome 6p21, whereas NCR3-412 polymorphism ( rs2736191 ) lying within this region was found to be associated with mild malaria.
Methods
Blood samples were taken from 188 Plasmodium falciparum malaria patients (76 mild malaria patients, 85 cerebral malaria patients, and 27 severe non-cerebral malaria patients). NCR3-412 ( rs2736191 ) was analysed by sequencing, and haematological parameters were measured. Finally, their association with clinical phenotypes was assessed.
Results
We evidenced an association of thrombocytopenia with both cerebral malaria and severe non-cerebral malaria, and of an association of high leukocyte count with cerebral malaria. Additionally, we found no association of NCR3-412 with either cerebral malaria, severe non-cerebral malaria, or severe malaria after grouping cerebral malaria and severe non-cerebral malaria patients.
Conclusions
Our results suggest that NCR3 genetic variation has no effect, or only a small effect on the occurrence of severe malaria, although it has been strongly associated with mild malaria. We discuss the biological meaning of these results. Besides, we confirmed the association of thrombocytopenia and high leukocyte count with severe malaria phenotypes.
... Upregulation of endothelial and plateletderived micro-particles with increased adhesiveness of endothelial cell-leukocyte-platelet interaction was shown in children with CM. In addition, platelet adhesion and aggregation are involved in cytoadhesion of PfIEs [12,13] and in modulating sequestration [14]. Furthermore, the severity of CM in children was correlated with the inhibition of ADAMTS13 and thus with an increase of abnormal circulating ultra large von Willebrand factor (ULVWF) multimers that cross-link platelets, induce thrombocytopaenia [5] and enhance obstruction of the brain microvasculature [15,16]. ...
Background:
Cerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection. A number of pathological findings have been correlated with pediatric CM including sequestration, platelet accumulation, petechial haemorrhage and retinopathy. However, the molecular mechanisms leading to death in CM are not yet fully understood.
Methods:
A shotgun plasma proteomic study was conducted using samples form 52 Gambian children with CM admitted to hospital. Based on clinical outcome, children were assigned to two groups: reversible and fatal CM. Label-free liquid chromatography-tandem mass spectrometry was used to identify and compare plasma proteins that were differentially regulated in children who recovered from CM and those who died. Candidate biomarkers were validated using enzyme immunoassays.
Results:
The plasma proteomic signature of children with CM identified 266 proteins differentially regulated in children with fatal CM. Proteins from the coagulation cascade were consistently decreased in fatal CM, whereas the plasma proteomic signature associated with fatal CM underscored the importance of endothelial activation, tissue damage, inflammation, haemolysis and glucose metabolism. The concentration of circulating proteasomes or PSMB9 in plasma was not significantly different in fatal CM when compared with survivors. Plasma PSMB9 concentration was higher in patients who presented with seizures and was significantly correlated with the number of seizures observed in patients with CM during admission.
Conclusions:
The results indicate that increased tissue damage and hypercoagulability may play an important role in fatal CM. The diagnostic value of this molecular signature to identify children at high risk of dying to optimize patient referral practices should be validated prospectively.
... The comparatively higher association affinity of CD36 and our lifetime measurements indicate that it is not necessary for high amounts of this receptor to be present to stabilize the binding of iRBCs. Furthermore, platelets have been shown to bind to endothelial cells and mediate binding of iRBCs, of which CD36 is one of the potential receptors involved 48,49 . Hence, it is likely that ICAM-1 is not solely responsible for all the sequestration in cerebral vasculature. ...
The adhesion of malaria infected red blood cells (iRBCs) to host endothelial receptors in the microvasculature, or cytoadhesion, is associated with severe disease pathology such as multiple organ failure and cerebral malaria. Malaria iRBCs have been shown to bind to several receptors, of which intercellular adhesion molecule 1 (ICAM-1) upregulation in brain microvasculature is the only one correlated to cerebral malaria. We utilize a biophysical approach to study the interactions between iRBCs and ICAM-1. At the single molecule level, force spectroscopy experiments reveal that ICAM-1 forms catch bond interactions with Plasmodium falciparum parasite iRBCs. Flow experiments are subsequently conducted to understand multiple bond behavior. Using a robust model that smoothly transitions between our single and multiple bond results, we conclusively demonstrate that the catch bond behavior persists even under flow conditions. The parameters extracted from these experimental results revealed that the rate of association of iRBC-ICAM-1 bonds are ten times lower than iRBC-CD36 (cluster of differentiation 36), a receptor that shows no upregulation in the brains of cerebral malaria patients. Yet, the dissociation rates are nearly the same for both iRBC-receptor interactions. Thus, our results suggest that ICAM-1 may not be the sole mediator responsible for cytoadhesion in the brain.
... Conversely, activated platelets can directly potentiate IE damage to the endothelium [119], and indirectly enhance the recruitment of both IEs and monocytes through the release of microparticles [120]. Platelets bind to the activated endothelium or to VWF fibers, thus creating a possible bridge for CD36-binding parasites in the microvasculature of organs with low expression of this receptor, such as the brain [121]. Collectively, these observations make a strong case for the potential pathological significance of altered coagulation in disease pathology. ...
Severe malaria due to Plasmodium falciparum infection causes nearly half a million deaths per year. The different symptomatology and disease manifestations among patients have hampered understanding of severe malaria pathology and complicated efforts to develop targeted disease interventions. Infected erythrocyte sequestration in the microvasculature plays a critical role in the development of severe disease, and there is increasing evidence that cytoadherent parasites interact with host factors to enhance the damage caused by the parasite. The recent discovery that parasite binding to endothelial protein C receptor (EPCR) is associated with severe disease has suggested new mechanisms of pathology and provided new avenues for severe malaria adjunctive therapy research.
... In addition to mechanical obstruction caused by PRBCs, a pronounced proinflammatory context with high plasma levels of cytokines, notably interferon-␥, TNF, and lymphotoxin, causes up-regulation of surface molecules on vascular cells, which then leads to enhanced interactions between various cell types within the vascular lumen (15). In vitro, platelets have been shown to bind to PRBCs (16,17) and to bridge PRBCs and brain endothelial cells, thus enhancing the binding of the former (18). Furthermore, platelets can have a direct cytotoxic effect on the monolayer of human brain endothelial cells (HBECs), an effect that is amplified by the presence of PRBCs (19). ...
... Several lines of evidence suggest a role of platelets in modulating endothelial adhesion phenotype and reorienting pRBCs for binding to endothelial cells in the brain microvasculature. Platelets may either provide adhesion receptors to vascular beds originally devoid of it and/or can hide receptors that are present (Wassmer et al. 2004). This in vitro study indicates that platelets can bring about pRBC CD36 binding to TNF activated CD36 deficient brain microvascular endothelial cells. ...
One of the most common central nervous system diseases in tropical countries is cerebral malaria (CM). Malaria is a common protozoan infection that is responsible for enormous worldwide mortality and economic burden on the society. Episodes of Plasmodium falciparum (Pf) caused CM may be lethal, while survivors are likely to suffer from persistent debilitating neurological deficits, especially common in children. In this review article, we have summarized the various symptoms and manifestations of CM in children and adults, and entailed the molecular basis of the disease. We have also emphasized how pathogenesis of the disease is effected by the parasite and host responses including blood brain barrier (BBB) disruption, endothelial cell activation and apoptosis, nitric oxide bioavailability, platelet activation and apoptosis, and neuroinflammation. Based on a few recent studies carried out in experimental mouse malaria models, we propose a basis for the neurological deficits and sequelae observed in human cerebral malaria, and summarize how existing drugs may improve prognosis in affected individuals.
... A subset of IRBCs also forms clumps or agglutinates to other IRBC [ 16 ] or forms rosettes by adhering to non-infected erythrocytes [ 17 ]. In addition to direct parasite adhesion to host receptors, platelets can act as a bridge between IRBC and endothelial cells, providing additional CD36 receptors for cytoadhesion [ 18 ]. IRBC adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family [ 19 -21 ]. ...
Plasmodium falciparum -infected red blood cells (IRBC) adhere to the endothelium via receptors expressed on the surface of vascular endothelial cells (EC) and sequester in the microvasculature of several organs. Sequestration is the primary step leading to complications related to the severity of malaria. In order to study this cytoadhesion phenomenon, IRBC in vitro binding assays have been developed using a monolayer of primary or transformed endothelial cells. Here we describe the methodology of an assay to inhibit the binding of IRBC on vascular endothelial cells under static adhesion conditions. Similar techniques could be used for conducting a binding inhibition assay under flow assay conditions using an appropriate device.
Cerebral malaria (CM) is a major cause of mortality in Plasmodium falciparum (Pf) infection and is associated with the sequestration of parasitised erythrocytes in the microvasculature of the host’s vital organs. Prompt diagnosis and treatment are key to a positive outcome in CM. However, current diagnostic tools remain inadequate to assess the degree of brain dysfunction associated with CM before the window for effective treatment closes. Several host and parasite factor-based biomarkers have been suggested as rapid diagnostic tools with potential for early CM diagnosis, however, no specific biomarker signature has been validated. Here, we provide an updated review on promising CM biomarker candidates and evaluate their applicability as point-of-care tools in malaria-endemic areas.
Infections with Plasmodium falciparum and Plasmodium vivax cause over 600,000 deaths each year, concentrated in Africa and in young children, but much of the world’s population remain at risk of infection. In this article, we review the latest developments in the immunogenicity and pathogenesis of malaria, with a particular focus on P. falciparum, the leading malaria killer. Pathogenic factors include parasite-derived toxins and variant surface antigens on infected erythrocytes that mediate sequestration in the deep vasculature. Host response to parasite toxins and to variant antigens is an important determinant of disease severity. Understanding how parasites sequester, and how antibody to variant antigens could prevent sequestration, may lead to new approaches to treat and prevent disease. Difficulties in malaria diagnosis, drug resistance, and specific challenges of treating P. vivax pose challenges to malaria elimination, but vaccines and other preventive strategies may offer improved disease control.
Cerebral malaria (CM) is one of the leading causes of death due to malaria. It is characterised by coma, presence of asexual parasites in blood smear, and absence of any other reason that can cause encephalopathy. The fatality rate for CM is high, and those who survive CM often experience long-term sequelae, including cognitive and motor dysfunctions. It is unclear how parasites sequestered in the lumen of endothelial cells of the blood–brain barrier (BBB), and localised breakdown of BBB can manifest gross physiological changes across the brain. The pathological changes associated with CM are mainly due to the dysregulation of inflammatory and coagulation pathways. Other factors like host and parasite genetics, transmission intensity, and the host’s immune status are likely to play a role in the development and progression of CM. This work focuses on the pathological mechanisms underlying CM. Insights from humans, mice, and in vitro studies have been summarised to present a cohesive understanding of molecular mechanisms involved in CM pathology.
Interactions between the immune and nervous systems are involved in many disease processes. Modulation of inflammation can provide an important opportunity to enhance regeneration within the central nervous system. This authoritative book defines the key cellular players in mounting an inflammatory response and highlights critical factors in the target organ that influence the nature of that response and its capacity either to damage or protect the brain. Several key clinical areas are highlighted – particularly autoimmune diseases of the nervous system including multiple sclerosis, as well as microbiological and traumatic challenges; the book therefore provides both a summary of the basic science background as well as practical, clinical-friendly guidelines to management. The book will be of interest to a wide range of physicians, including neurologists, neurosurgeons, neurorehabilitationists, infectious disease physicians, and clinical neuroscientists, as well as neuroscientists and immunologists.
The methods presented in this chapter describe how to perform ex vivo clumping and in vitro bridging assays in the context of cerebral malaria. Both the protocols are detailed, and emphasis is made on how to prepare platelet suspensions suitable to each technique, including description of specific buffers and reagents to minimize the risk of aggregation while maintaining the platelet properties.
Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling.
Riassunto
Con quasi 500 000 morti ogni anno in zone di endemia, la malaria grave è una priorità di salute pubblica a livello mondiale. In Francia e nei paesi non endemici, la malaria grave di importazione resta un’infezione potenzialmente letale, nonostante un livello ottimale di cura. La fisiopatologia ruota attorno a diversi meccanismi, il più importante dei quali è la citoadesione dei globuli rossi parassitati all’endotelio vascolare. La diagnosi di malaria grave si basa sulla dimostrazione di Plasmodium falciparum (o, molto più raramente, delle altre quattro specie) nel sangue e sulla presenza di almeno un criterio clinico o laboratoristico di gravità, come definito dall’Organizzazione Mondiale della Sanità nel 2014 e adattato alla malaria grave di importazione in Francia nelle linee guida di pratica clinica riviste nel 2017. Dopo l’era del chinino, il trattamento antinfettivo per la malaria grave di importazione è, ora, basato sull’artesunato per via endovenosa negli adulti, nei bambini e nelle donne gravide. A livello sintomatico, soprattutto durante nelle forme più gravi, la gestione in rianimazione delle insufficienze d’organo è fondamentale e presenta alcune specificità. A tutt’oggi, nessun trattamento adiuvante (inclusa l’exsanguinotrasfusione) ha dimostrato la sua efficacia nella pratica clinica. Più a monte, l’impatto della malaria grave potrebbe essere ridotto migliorando la prevenzione (chemioprofilassi, informazioni per il pubblico in generale) e migliorando la diagnosi e il trattamento nella fase di malaria semplice. Infine, l’elaborazione di un vaccino progredisce, ma la sua efficacia resta ancora modesta.
Resumen
Con cerca de 500.000 fallecimientos anuales en zonas de endemia, el paludismo grave es una prioridad de salud pública a escala mundial. En los países no endémicos, el paludismo grave importado sigue siendo una infección potencialmente mortal a pesar de una atención óptima. La fisiopatología se articula alrededor de varios mecanismos, el más importante de los cuales es la citoadherencia de los hematíes parasitados al endotelio vascular. El diagnóstico de paludismo grave se basa en la detección de Plasmodium falciparum (o mucho más raramente de las otras cuatro especies) en la sangre y en la presencia de al menos un criterio clínico o biológico de gravedad, como definió la Organización Mundial de la Salud en 2014, y se adaptó al paludismo grave importado en Francia en las recomendaciones para la práctica clínica revisadas en 2017. Después de la era de la quinina, el tratamiento antiinfeccioso del paludismo grave importado se basa ahora en el artesunato intravenoso en el adulto, el niño y la mujer embarazada. En el plano sintomático, en particular en las formas más graves, el tratamiento en reanimación de las insuficiencias orgánicas es primordial y comporta algunas especificidades. En la actualidad, ningún tratamiento coadyuvante (incluida la exanguinotransfusión) ha demostrado su eficacia en la práctica clínica. Previamente, se podría disminuir el impacto del paludismo grave mediante la mejora de la prevención (quimioprofilaxis, información al gran público) y mediante una mejora del diagnóstico y el tratamiento en estado de acceso palúdico simple. Finalmente, la elaboración de una vacuna avanza, pero su eficacia todavía es modesta.
Objective: To observe and compare placental histology for hypoxic changes in diet plus exercise alone versus diet plus exercise and insulin treatment in patients with Gestational Diabetes Mellitus (GDM).. Materials and Methods: After written informed consent, 30 patients were diagnosed to have GDM with RBS between 126-129mg/dl were given diet control plus exercise therapy (Group A). 39 GDM patients with RBS greater than 130 mg/dl were kept on diet plus exercise and insulin (Group B). After delivery placentae were collected from 25 patients in each group. Histological slides of placentae were prepared and parameters of hypoxia such as villous immaturity, villous fibrinoid necrosis, syncytial knots, chorangiosis, calcification etc. were observed and compared between the two groups using light microscope. Results were evaluated by SPSS 16 using student t-test and chi square test. Results: Statistically non-significant results were obtained for the hypoxic parameters. However numerically more calcification was found in Group A while villous immaturity, villous fibrinoid necrosis and syncytial knots were present more in Group B. Conclusion: Histological examination of placentae showed presence of hypoxic features in both Group A and Group B patients with more propensities in diet plus exercise and insulin treated GDM group.
Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson’s correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406–651] vs. 1029 [732–1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.
Sequestration of P. falciparum-parasitized red blood cells (PRBC) in vital organs, the brain in particular, is a common pathological finding of cerebral malaria (CM). Ginger (Zingiber officinale) has several pharmacological Effects; such as effects on the gastrointestinal tract, antimicrobial effects, effects on cardiovascular system, effect on blood pressure and effect on blood clotting. Therefore, this study aimed to investigate the ability of Ginger to safe C57BL/6-WT mice from cerebral malaria caused by Plasmodium berghei ANKA. Infected C57BL/6-Wild type mice with Plasmodium berghei ANKA have been used in this study. Two mice groups 6 mice in each: 1) Ginger group which treated iv with 150 mg/kg of Ginger extract and 2) Control group which injected iv with normal saline. The outcome results in this study showed a high ability of Ginger extract to safe C57BL/6-WT mice from the cerebral malaria (P-Value= 0.023.), with no antimalarial activity. In a conclusion, the Ginger extract has the ability to save 100% of the infected C57BL/6-Wild type mice during cerebral malaria time points. More studies are also required on the ginger and its constituents in reducing circulated TNF and LT-α and to reduce platelets and RBCs cytoadherence.
Key Points
Nbeal2 deficiency leads to significantly reduced lung and brain pathology and enhanced survival in a mouse model of malaria. Both antibody-dependent and antibody-independent platelet depletion in mice recapitulate the findings observed in Nbeal2−/− mice.
Sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature is a hallmark of cerebral malaria (CM). Using a microchannel flow adhesion assay with TNF-activated primary human microvascular endothelial cells, we demonstrate that IE isolated from Malawian paediatric CM cases showed increased binding to brain microvascular endothelial cells compared to IE from uncomplicated malaria (UM) cases. Further, UM isolates showed significantly greater adhesion to dermal than to brain microvascular endothelial cells. The major mediator of parasite adhesion is P. falciparum erythrocyte membrane protein 1, encoded by var genes. Higher levels of var gene transcripts predicted to bind host endothelial protein C receptor (EPCR) and ICAM-1 were detected in CM isolates. These data provide further evidence for differential tissue binding in severe and uncomplicated malaria syndromes, and give additional support to the hypothesis that CM pathology is based on increased cytoadherence of IE in the brain microvasculature. © 2019 The Authors. Published under the terms of the CC BY 4.0 license
Cerebral malaria (CM) is an acute encephalopathy caused by the malaria parasite Plasmodium falciparum, which develops in a small minority of infected patients and is responsible for the majority of deaths in African children. Despite decades of research on CM, the pathogenic mechanisms are still relatively poorly defined. Nevertheless, many studies in recent years, using a combination of animal models, in vitro cell culture work, and human patients, provide significant insight into the pathologic mechanisms leading to CM. In this review, we summarize recent findings from mouse models and human studies on the pathogenesis of CM, understanding of which may enable development of novel therapeutic approaches.
Thrombocytopenia is a well-known manifestation of acute tropical infectious diseases. The role of platelets in infections has received much attention recently because of their emerging activities in modulation of inflammatory responses, host defense, and vascular integrity. However, while many studies have addressed thrombocytopenia in tropical infections, abnormalities in platelet function have been largely overlooked. This is an important research gap, as platelet dysfunction may contribute to the bleeding tendency that characterizes some tropical infections. The development of novel platelet function assays that can be used in thrombocytopenic conditions (e.g., flow cytometry assays) has contributed to important new insights in recent years. In this review, the importance of platelets in tropical infections is discussed with special emphasis on the underlying mechanisms and consequences of thrombocytopenia and platelet dysfunction in these infections. Special attention is paid to malaria, a disease characterized by microvascular obstruction in which bleeding is rare, and to infections in which bleeding is common, such as dengue, other viral hemorrhagic fevers, and the bacterial infection leptospirosis. Given the importance of platelet function abnormalities in these infections, the development of affordable assays for monitoring of platelet function in low-resource countries, as well as pharmacologic interventions to prevent or reverse platelet function abnormalities, might improve clinical care and the prognosis of these infections.
Purpose:
The objective of this study was to study the altered proteome in the frontal lobe of patients with CM. Unbiased analysis of differentially abundant proteins could lead to identification of host responses against P. falciparum infection, which will aid in better understanding of the molecular mechanism of pathophysiology in CM.
Experimental design:
We employed TMT-based quantitative proteomic analysis using high-resolution mass spectrometry. In brief, proteins were isolated from frontal lobe samples, which were collected at autopsy from three cases of CM and three control subjects. Equal amounts of protein from each case were digested using trypsin and labeled with different TMT reagents. The pooled sample was fractionated using strong cation exchange chromatography and analyzed on Orbitrap Fusion in triplicates. For accurate quantitation of peptides, the samples were analyzed in MS3 mode. The data was searched against a combined database of human and P. falciparum proteins using Sequest and Mascot search engines.
Results:
A total of 4,174 proteins were identified, of which, 107 were found to be differentially abundant in the test samples with significant p-value (<0.05). Proteins associated with biological processes such as innate immune response, complement system, coagulation and platelet activation were found to be elevated in CM cases. In contrast, proteins associated with myelination, oxidative phosphorylation, regulation of reactive oxygen species, sodium and calcium ions transport were found to be depleted in response to CM. In addition, we also identified three P. falciparum proteins exclusively in CM brain samples.
Conclusions and clinical relevance:
Our study signifies neuronal assault due to axonal injury, altered sodium and calcium ion channels, deregulated inflammation and demyelination as a part of host response to CM. Enhanced oxidative stress, repressed oxidative phosphorylation and demyelination of axons may contribute to the severity of the disease. Further validation of these results on a large cohort can provide leads in the development of neuroprotective therapies for CM. This article is protected by copyright. All rights reserved.
Infections continue to cause a high incidence of mortality and morbidity in resource-poor nations. Although antimicrobial therapy has aided mostly in dealing with the pathogenic micro-organisms themselves, the collateral damage caused by the infections continue to cause many deaths. Intensive care support and manipulation of the hosts' abnormal response to the infection have helped to improve mortality in well-resourced countries. But, in those areas with limited resources, this is not yet the case and simpler methods of diagnosis and interventions are required. Thrombocytopenia is one of the most common manifestations in all these infections and may be used as an easily available prognostic indicator and marker for the severity of the infections. In this review, the relevance of platelets in infections in general, and specifically to tropical infections, malaria, and viral haemorrhagic fevers in the emerging countries is discussed. Better understanding of the pathophysiology and the role of platelets in particular in such conditions is likely to translate into better patient care and thus reduce morbidity and mortality.
Nitric oxide (NO) is widely accepted as a central regulator of vascular tone and a vast array of other cardiovascular signaling mechanisms. An emerging player in these mechanisms is hemoglobin (Hb), an erythrocytic protein that serves as the archetypical model for an allosteric protein. Specifically, red blood cells (RBC) are suggested to be integral in matching blood flow to tissue oxygen demands. The mechanisms proposed involve the ability of Hb to sense changes in oxygen concentrations and coupling this process to modulating vascular NO levels. The molecular basis of these mechanisms remains under investigation, but is clearly diverse and discussed in this article from the basis of the blood flow responses to hypoxia. Another emerging theme in RBC biology is the role of these cells during inflammatory disease in which disease processes promote the interaction of vascular NO and the RBC. This is exemplified in hemolytic diseases, in which released Hb has drastic affects on vascular homeostasis mechanisms. Additionally, it is becoming evident that RBC express numerous molecules that mediate interactions with the extracellular matrix and cellular mediators of inflammation. The functional implications for such interactions remain unclear but highlight potential roles of the RBC in modulating inflammatory disease.
Severe Plasmodium falciparum malaria remains a leading cause of mortality, particularly in sub-Saharan Africa where it accounts for up to 1 million deaths per annum. In spite of the significant mortality and morbidity associated with cerebral malaria (CM), the molecular mechanisms involved in the pathophysiology of severe malaria remain surprisingly poorly understood. Previous studies have demonstrated that sequestration of P. falciparum-infected erythrocytes within the microvasculature of the brain plays a key role in the development of CM. In addition, there is convincing evidence that both EC activation and platelets play critical roles in the modulating the pathogenesis of severe P. falciparum malaria. In this review, we provide an overview of recent studies that have identified novel roles through which haemostatic dysfunction may directly influence malaria pathogenesis In particular, we focus on emerging data suggesting that von Willebrand factor, coagulation cascade activation and dysfunction of the protein C pathway may be of specific importance in this context. These collective insights underscore a growing appreciation of the important, but poorly understood, role of haemostatic dysfunction in malaria progression and importantly, illuminate potential approaches for novel therapeutic strategies. Given that the mortality rate associated with CM remains in the order of 20% despite the availability of effective anti-malarial therapy, development of adjunctive therapies that can attenuate CM progression clearly represents a major unmet need. These emerging data are thus not of only of basic scientific interest, but also of direct clinical significance.
Platelets are highly specialized for inflammatory responses, in addition to their well-known specializations as effector cells in hemostasis. Experimental and clinical observations demonstrate that platelets have activities that span the immune continuum, and that they have cellular and molecular capabilities that allow them to participate across the spectrum of physiologic and pathologic inflammation. Some of these biologic capabilities have been discovered only recently. In many cases, platelet effector mechanisms link inflammation and hemostasis. There is substantial evidence that platelets have critical activities in a variety of inflammatory diseases and syndromes, based on clinical studies, in vitro analysis, and pre-clinical models.
There are nearly 1 million malaria deaths yearly, primarily in sub-Saharan African children. Cerebral malaria (CM), marked by coma and sequestered malaria parasites in brain blood vessels, causes half of these deaths, although the mechanisms causing coma and death are uncertain. Sub-Saharan Africa has a high HIV prevalence, with 3 million HIV-infected (HIV⁺) children, but the effects of HIV on CM pathogenesis and mortality are unknown. In a study of pediatric CM in Malawi, HIV prevalence was high and CM-attributed mortality was higher in HIV⁺ than in HIV-uninfected children. Brain pathology in children with fatal CM was notable not only for sequestered malaria parasites but also for intravascular accumulations of monocytes and platelets that were more severe in HIV⁺ children. Our findings raise the possibility that HIV⁺ children at risk for malaria may benefit from targeted malaria prophylaxis and that adjunctive treatments targeting inflammation and/or coagulation may improve CM outcomes.
The clinical complications associated with severe and cerebral malaria occur as a result of the intravascular mechanical obstruction of erythrocytes infected with the asexual stages of the parasite, Plasmodium falciparum. We now report that a primary P. falciparum-infected erythrocyte (parasitized red blood cell [PRBC]) isolate from a patient with severe complicated malaria binds to cytokine-induced human vascular endothelial cells, and that this adhesion is in part mediated by endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). PRBC binding to tumor necrosis factor alpha (TNF-alpha)-activated human vascular endothelial cells is partially inhibited by antibodies to ELAM-1 and ICAM-1 and the inhibitory effects of these antibodies is additive. PRBCs selected in vitro by sequential panning on purified adhesion molecules bind concurrently to recombinant soluble ELAM-1 and VCAM-1, and to two previously identified endothelial cell receptors for PRBCs, ICAM-1, and CD36. Post-mortem brain tissue from patients who died from cerebral malaria expressed multiple cell adhesion molecules including ELAM-1 and VCAM-1 on cerebral microvascular endothelium not expressed in brains of individuals who died from other causes. These results ascribe novel pathological functions for both ELAM-1 and VCAM-1 and may help delineate alternative adhesion pathways PRBCs use to modify malaria pathology.
The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P. falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.
Cultured human umbilical vein endothelial cells (EC) constitutively express a low level of CD40 antigen as detected by monoclonal antibody binding and fluorescence flow cytometric quantitation. The level of expression on EC is increased about 3-fold following 24 h treatment with optimal concentrations of tumor necrosis factor, interleukin 1, interferon beta, or interferon gamma; both interferons show greater than additive induction of CD40 when combined with tumor necrosis factor or interleukin 1. Expression of CD40 increases within 8 h of cytokine treatment and continues to increase through 72 h. A trimeric form of recombinant murine CD40 ligand acts on human EC to increase expression of leukocyte adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. CD40 may be detected immunocytochemically on human microvascular EC in normal skin. We conclude that endothelial CD40 may play a role as a signaling receptor in the development of T-cell-mediated inflammatory reactions.
Pathogenic mechanisms of brain microvascular injury were studied in an experimental model of cerebral malaria (CM). The lesion, leading to perivascular microhemorrhages, is due to cytokine overproduction, and is associated with the sequestration of macrophages and parasitized erythrocytes in cerebral venules. In this in vivo model, we demonstrate that platelets are critical effectors of the neurovascular injury. First, electron microscopy indicated that during CM platelets adhere to and probably damage brain endothelial cells. Second, radiolabelled platelet distribution studies indicated that platelets sequestered in the brain and lung vasculature during CM. Non-cerebral malaria was not associated with cerebral sequestration of platelets. Third, in vivo treatment with a mAb to LFA-1 (which is expressed on platelets) selectively abrogated the cerebral sequestration of platelets, and this correlated with prevention of CM. Fourth, malaria-infected animals rendered thrombocytopenic were significantly protected against CM, further indicating that platelets are central to the pathogenesis of CM. Thus, a CD11a-dependent interaction between platelets and endothelial cells appears pivotal to microvascular damage. These data suggest a novel mechanism of action for anti-LFA-1 mAb in vivo and illustrate an unexpected role of platelets, in addition to monocytes, in vascular pathology.
Adhesion of parasitized red blood cells (PRBCs) to microvascular endothelial cells (ECs) is a distinctive feature of Plasmodium falciparum malaria and is a central event in the development of life-threatening complications such as cerebral malaria. PRBCs adhere to several EC-expressed molecules in vitro, but the relative importance of these interactions in vivo remains unclear. Chondroitin sulfate A (CSA) is the most recent EC surface-associated molecule to be implicated in the adhesive process. Accordingly, we have studied adhesion of PRBCs to CSA in vitro using a parallel-plate flow chamber. Under controlled flow conditions, PRBCs adhered to CSA in a concentration-dependent manner at wall-shear stresses up to 0.2 Pa, a value that is within the physiological range for venules. Once adhered, PRBCs remained stationary (rather than rolling) and continued to remain stationary even when the wall-shear stress was raised to supravenular levels. The adhesive interaction was strong and a proportion of adherent PRBCs could withstand detachment at stresses up to 2.5 Pa. Soluble CSA at pharmacological concentrations prevented adhesion of flowing PRBCs in a concentration-dependent manner but failed to reverse established adhesion. Adhesion of PRBCs to CSA could contribute to the pathogenesis of malaria, and soluble CSA may have a useful therapeutic effect.
Late stages of Plasmodium falciparum-infected erythrocytes (IRBCs) frequently sequester in the placentas of pregnant women, a phenomenon associated with low birth weight of the offspring. To investigate the physiological mechanism of this sequestration, we developed an in vitro assay for studying the cytoadherence of IRBCs to cultured term human trophoblasts. The capacity for binding to the syncytiotrophoblast varied greatly among P. falciparum isolates and was mediated by intercellular adhesion molecule 1 (ICAM-1), as binding was totally inhibited by 84H10, a monoclonal antibody specific for ICAM-1. Binding of the P. falciparum line RP5 to the syncytiotrophoblast involves chondroitin-4-sulfate (CSA), as this binding was dramatically impaired by addition of free CSA to the binding medium or by preincubation of the syncytiotrophoblast with chondroitinase ABC. ICAM-1 and CSA were visualized on the syncytiotrophoblast by immunofluorescence, while CD36, E-selectin, and vascular cell adhesion molecule 1 were not expressed even on tumor necrosis factor alpha (TNF-alpha)-stimulated syncytiotrophoblast tissue, and monoclonal antibodies against these cell adhesion molecules did not inhibit cytoadherence. ICAM-1 expression and cytoadherence of wild isolates was upregulated by TNF-alpha, a cytokine that can be secreted by the numerous mononuclear phagocytes present in malaria-infected placentas. These results suggest that cytoadherence may be involved in the placental sequestration and broaden the understanding of the physiopathology of the malaria-infected placenta.
Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells.
To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1.
The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA.
In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.
Various molecules expressed on the surface of platelets have been shown to mediate the protective or deleterious role of these cells in immuno-inflammatory mechanisms. Increasing evidence points to the involvement of the cell adhesion molecules, gpIIb-IIIa, P-selectin, CD31, LFA-1, and CD36 in the interaction between platelets and endothelial cells as well as other cell types. The possible role of these molecules in the ability of platelets to support endothelium and to protect against tumour necrosis factor mediated cytolysis or parasitic invasion are reviewed. The involvement of platelets as effectors of tissue damage in cerebral malaria, lipopolysaccharide induced pathology, and pulmonary fibrosis is also discussed. This has then been extended to include the intercellular mechanisms underpinning their pathogenic role in metastasis, transplant rejection, stroke, brain hypoxia, and related conditions. A better understanding of the complex regulation and hierarchical organisation of these various platelet adhesion molecules may prove useful in the development of new approaches to the treatment of such diseases.
Tumor necrosis factor-alpha (TNF) is known to be an important mediator in the pathogenesis of several inflammatory diseases. Vascular endothelial cells represent a major target of TNF effects. Platelet sequestration has been found in brain microvessels during experimental cerebral malaria and lung in experimental pulmonary fibrosis, implying that it may participate in TNF-dependent microvascular pathology. In this study, we investigated the mechanisms of platelet-endothelial interaction, using co-cultures between platelets and TNF-activated mouse brain microvascular endothelial cells (MVECs). Adhesion and fusion of platelets to MVECs was evidenced by electron microscopy, dye transfer, and flow cytometry. It was induced by TNF and interferon-gamma and depended on LFA-1 expressed on the platelet surface and ICAM-1 expressed on MVECs. The adhesion and fusion also led to the transfer of platelet markers on the MVEC surface, rendering these more adherent for leukocytes, and to an enhanced MVEC sensitivity to TNF-induced injury. These results suggest that platelets can participate in TNF-induced microvascular pathology.
Members of the Plasmodium falciparum var gene family encode clonally variant adhesins, which play an important role in the pathogenicity of tropical malaria. Here we employ a selective panning protocol to generate isogenic P.falciparum populations with defined adhesive phenotypes for CD36, ICAM-1 and CSA, expressing single and distinct var gene variants. This technique has established the framework for examining var gene expression, its regulation and switching. It was found that var gene switching occurs in situ. Ubiquitous transcription of all var gene variants appears to occur in early ring stages. However, var gene expression is tightly regulated in trophozoites and is exerted through a silencing mechanism. Transcriptional control is mutually exclusive in parasites that express defined adhesive phenotypes. In situ var gene switching is apparently mediated at the level of transcriptional initiation, as demonstrated by nuclear run-on analyses. Our results suggest that an epigenetic mechanism(s) is involved in var gene regulation.
The cytoadhesion of Plasmodium falciparum laboratory strains and clones to Saimiri brain microvascular endothelial cells (SBEC 17), with chondroitin-4-sulfate (CSA) as the only adhesion receptor, was tested. Only one strain had significant cytoadhesion. However, CSA-specific infected erythrocytes (IRBCs) were detected in all strains after selection of a CSA-specific subpopulation by culturing the few adherent IRBCs. This demonstrates the lack of sensitivity of cytoadhesion microassays for detecting small quantities of CSA-specific IRBCs in cultures or field isolates. Cytoadhesion to CSA is maximal at 24 h of the cycle and decreases with the onset of schizogony, reaching a minimum just before reinvasion. This fluctuation must be taken into account in comparisons of the cytoadhesion of different strains or isolates. The minimum size of CSA for active inhibition was 4 kDa, and a mass of 9 kDa was required for inhibition similar to that obtained with the 50-kDa CSA. In contrast to cytoadhesion to CSA, which is pH independent or maximal at physiological pH (depending on the target endothelial cells), adhesion to CD36 and intercellular adhesion molecule 1 was pH dependent, requiring acidic conditions to be maximal in all cases. Cytoadhesion to CSA may trigger the occlusion of microvessels and cause the acidosis necessary for the other receptors to be fully efficient. If this key role in the mechanisms of sequestration were to be confirmed in vivo, prevalence studies of the CSA cytoadhesion phenotype would have to be reevaluated, because simple cytoadhesion assays do not detect CSA-specific parasites present in very low numbers, and these parasites might then be undetected in the peripheral blood but present in organs in which sequestration occurs, such as the placenta (M. Fried and P. E. Duffy, Science 272:1502-1504, 1996).
Plasmodium falciparum malaria during pregnancy is an important cause of maternal and infant morbidity and mortality. Accumulation of large numbers
of P. falciparum—infected erythrocytes in the maternal blood spaces of the placenta may be mediated by adhesion of infected erythrocytes to molecules
presented on the syncytiotrophoblast surface. In this study, isolates from placentas and peripheral blood of infected pregnant
women and from children were tested for binding to purified receptors and for agglutination with adult sera. Results suggest
that adhesion to chondroitin sulfate A may be involved in placental parasite sequestration in most cases, but other factors
are also likely to be important. Agglutination assay results suggest that parasites infecting pregnant women are antigenically
distinct from those common in childhood disease. The prevalence of agglutinating antibodies to pregnancy isolates was generally
low, but it was highest in multigravidae who are likely to have had the greatest exposure.
Malaria during the first pregnancy causes a high rate of fetal and neonatal death. The decreasing susceptibility during subsequent pregnancies correlates with acquisition of antibodies that block binding of infected red cells to chondroitin sulfate A (CSA), a receptor for parasites in the placenta. Here we identify a domain within a particular Plasmodium falciparum erythrocyte membrane protein 1 that binds CSA. We cloned a var gene expressed in CSA-binding parasitized red blood cells (PRBCs). The gene had eight receptor-like domains, each of which was expressed on the surface of Chinese hamster ovary cells and was tested for CSA binding. CSA linked to biotin used as a probe demonstrated that two Duffy-binding-like (DBL) domains (DBL3 and DBL7) bound CSA. DBL7, but not DBL3, also bound chondroitin sulfate C (CSC) linked to biotin, a negatively charged sugar that does not support PRBC adhesion. Furthermore, CSA, but not CSC, blocked the interaction with DBL3; both CSA and CSC blocked binding to DBL7. Thus, only the DBL3 domain displays the same binding specificity as PRBCs. Because protective antibodies present after pregnancy block binding to CSA of parasites from different parts of the world, DBL-3, although variant, may induce cross-reactive immunity that will protect pregnant women and their fetuses.
We performed ex vivo experiments with Plasmodium falciparum-infected human placentas from primi- and multigravida women from Cameroon. All women, independent of their gravida status, had anti-chondroitin sulfate A (CSA) adhesion antibodies which cross-reacted with heterologous strains, such as FCR3 and Palo-Alto(FUP)1, which were selected for CSA binding. These antibodies, directed against the surface of infected erythrocytes obtained by flushing with CSA (IRBC(CSA)), were restricted to the immunoglobulin G3 isotypes. Massive desequestration of parasites was achieved with soluble CSA but not with anti-ICAM-1 and anti-CD36 monoclonal antibodies. All of the CSA-flushed parasites were analyzed immediately by using in vitro assays of binding to Saimiri brain endothelial cells (SBEC) expressing various adhesion receptors. Parasites derived from all six placentas displayed the CSA adhesion phenotype. However, only partial inhibition of adhesion was observed in the presence of soluble CSA or when Sc1D SBEC were treated with chondroitinase ABC. These results suggest that an additional adhesive molecule of IRBC(CSA) which binds to an unidentified receptor is present in the placenta. This new phenotype was lost once the parasites adapted to in vitro culture. We observed additional differences in the CSA adhesion phenotype between placental parasites and in vitro-cultured parasites panned on endothelial cells carrying CSA. The minimum size of fractionated CSA required for a significant inhibition of placental IRBC(CSA) adhesion to Sc1D cells was 1 to 2 kDa, which contrasts with the 4-kDa size necessary to reach equivalent levels of inhibition with panned IRBC(CSA) of this phenotype. All placental IRBC(CSA) cytoadhered to Sc17 SBEC, which express only the CSA receptor. Panning of IRBC(CSA) on these cells resulted in a significant quantitative increase of IRBC cytoadhering to the CSA of Sc1D cells but did not change their capacity for adhesion to CSA on normal placenta cryosections. Our results indicate that the CSA binding phenotype is heterogeneous and that several distinct genes may encode P. falciparum-CSA ligands with distinct binding properties.
Using an in vitro model of human lung endothelial cells, we studied different characteristics of Plasmodium falciparum isolates as potential factors for malaria severity in 2 Thai patient groups: 27 with complicated malaria and 42 with uncomplicated malaria. In regard to binding properties, no association existed between cytoadherence and rosette phenotypes (P = 0.1) and hypothrombocytemia increased the cytoadherence level (P = 0.007). Cytoadherence was significantly associated with malaria severity (P = 0.05) in contrast to rosette formation (P = 0.9). Intercellular adhesion molecule-1 and chondroitin-4-sulfate were major receptors of cytoadherence in those with complicated malaria compared with those with uncomplicated malaria (P < 10(-4)). Chondroitin-4-sulfate could act as a putative receptor for malaria complications in non-pregnant women. CD36 was the main receptor in patients with uncomplicated malaria (P < 10(-3)). Vascular cell adhesion molecule-1 and E-selectin played a minor role in 2 groups (P = 0.6). Qinghaosu derivatives were more efficient than other antimalarial drugs, but a positive correlation was observed between the 50% inhibitory concentrations of halofantrine and quinine and the number of adhesive parasitized red blood cells, suggesting their influence on cytoadherence.
Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.
Malaria still is a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria, remains incompletely understood. Experimental models represent useful tools to better understand the mechanisms of this syndrome. Here, data generated by several models are reviewed both in vivo and in vitro; we propose that some pathogenic mechanisms, drawn from data obtained from experiments in a mouse model, may be instrumental in humans. In particular, tumor necrosis factor (TNF) receptor 2 is involved in this syndrome, implying that the transmembrane form of TNF may be more important than the soluble form of the cytokine. It has also been shown that in addition to differences in immune responsiveness between genetically resistant and susceptible mice, there are marked differences at the level of the target cell of the lesion, namely, the brain endothelial cell. In murine cerebral malaria, a paradoxical role of platelets has been proposed. Indeed, platelets appear to be pathogenic rather than protective in inflammatory conditions because they can potentiate the deleterious effects of TNF. More recently, it has been shown that interactions among platelets, leukocytes, and endothelial cells have phenotypic and functional consequences for the endothelial cells. A better understanding of these complex interactions leading to vascular injury will help improve the outcome of cerebral malaria.
Although chondroitin-4-sulfate (CSA) is expressed throughout the microvasculature and CSA-binding infected erythrocytes (IECSA) cytoadhere to lung and brain endothelial cells and sequester in male Saimiri sciureus this phenotype seems to be dependent on the presence of a placenta to develop. This contradiction was investigated by modeling
the interactions and cytoadhesion parameters in the microvasculature. Mixtures of IEs interacting with CSA, CD36, or intercellular
adhesion molecule 1 were incubated with endothelial cells expressing the corresponding receptors, at physiological pH, under
flow conditions. By use of suspensions composed of equal proportions of the phenotypes, cytoadhesion of ∼10 times as many
IECSA as of any other IE tested was observed. Adherent IECSA resisted microvascular wall shear stresses 3–15 times more effectively than did the others. These results, which require
confirmation with field isolates, demonstrate that the CSA phenotype is competitive and are consistent with this phenotype
initiating microvessel occlusion and with CSA-mediated sequestration in microvessel conditions
The pathogenesis of fatal cerebral malaria (CM) is not well understood, in part because data from patients in whom a clinical
diagnosis was established prior to death are rare. In a murine CM model, platelets accumulate in brain microvasculature, and
antiplatelet therapy can improve outcome. We determined whether platelets are also found in cerebral vessels in human CM,
and we performed immunohistopathology for platelet-specific glycoprotein, GPIIb-IIIa, on tissue from multiple brain sites
in Malawian children whose fatal illness was severe malarial anemia, CM, or nonmalarial encephalopathy. Platelets were observed
in 3 locations within microvessels: between malaria pigment and leukocytes, associated with malaria pigment, or alone. The
mean surface area of platelet staining and the proportion of vessels showing platelet accumulation were significantly higher
in patients with CM than in those without it. Platelet accumulation occurs in the microvasculature of patients with CM and
may play a role in the pathogenesis of the disease
Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.
We analyzed the role of adhesion molecules in the pathogenesis of experimental cerebral malaria (ECM), since tumor necrosis factor (TNF) plays a major role in this condition and has been shown to up-regulate in vitro expression of cell adhesion molecules (CAM), particularly intercellular CAM-1 (ICAM-1). We found increased expression of ICAM-1 on brain endothelial cells from mice with ECM. Treatment with monoclonal antibodies (mAb) directed against leukocyte function-antigen 1 (LFA-1, the ligand of ICAM-1) on days 6, 8 and 10 almost totally prevented ECM, while decreasing blood TNF levels. To exclude the possibility that the effects of anti-LFA-1 mAb resulted from an even partial inhibition of TNF overproduction, mice with signs of imminent death (hypothermia and neurologic defects) were treated with the anti-LFA-1 mAb, with dramatically protective effect. In contrast, injection of anti-ICAM-1 mAb on day 6 caused rapid death, while it was innocuous in normal mice. An mAb directed against complement receptor type 3 (CR3) was ineffective, as were injections of soluble human ICAM-1. These results suggest that adhesion of LFA-1+ cells to endothelial cells, stimulated by TNF to express high levels of ICAM-1, is critical in the pathogenesis of ECM. Emergency therapy at interfering with cytoadherence could be considered in the treatment of cerebral malaria in man, in which high blood TNF levels are also observed.
Adhesion of parasitized erythrocytes to microvascular endothelium is a central event in the pathogenesis of severe falciparum malaria. We have characterized the adhesion of flowing parasitized red blood cells to three of the known endothelial receptors coated on plastic surfaces (CD36, intercellular adhesion molecule-1 (ICAM-1) and thrombospondin (TSP)), and also to cells bearing these receptors (human umbilical vein endothelial cells (HUVEC) and platelets). All of the surfaces could mediate adhesion at wall shear stress within the physiological range. The great majority of adherent parasitized cells formed rolling rather than static attachments to HUVEC and ICAM-1, whereas static attachments predominated for platelets, CD36 and TSP. Studies with monoclonal antibodies verified that binding the HUVEC was mainly via ICAM-1, and to platelets via CD36. Adhesion via ICAM-1 was least sensitive to increasing wall shear stress, but absolute efficiency of adhesion was greatest for CD36, followed by ICAM-1, and least for TSP. TSP did not give long-lasting adhesion under flow, whereas cells remained adherent to CD36 or ICAM-1. We propose that the different receptors may have complementary roles in modulating adhesion in microvessels. Initial interaction at high wall shear stress may be of a rolling type, mediated by ICAM-1 or other receptors, with immobilization and stabilization occurring via CD36 and/or TSP.
The human malaria parasite Plasmodium falciparum evades host immunity by varying the antigenic and adhesive character of infected erythrocytes. We describe a large and extremely diverse family of P. falciparum genes (var) that encode 200-350 kDa proteins having the expected properties of antigenically variant adhesion molecules. Predicted amino acid sequences of var genes show a variable extracellular segment with domains having receptor-binding features, a transmembrane sequence, and a terminal segment that is a probable submembrane anchor. There are 50-150 var genes on multiple parasite chromosomes, and some are in clustered arrangements. var probes detect two classes of transcripts in steady-state RNA: 7-9 kb var transcripts, and an unusual family of 1.8-2.4 kb transcripts that may be involved in expression or rearrangements of var genes.
The adhesion of parasitized red blood cells (PRBC) to the endothelium (sequestration) may contribute to the pathogenic events in severe human malaria caused by P. falciparum. However, the factors involved in the pathophysiology, especially cerebral malaria are poorly understood. Previously, we have shown that the squirrel monkey Saimiri sciureus is a potential model for human cerebral malaria. In this paper we describe five stable clones of endothelial cell lines isolated immediately postmortem from different regions of the brain of Saimiri monkeys. The endothelial cell characteristics of these clones were confirmed by analyzing their ultrastructural aspects by transmission electron microscopy and by immunodetection of various endothelial cell markers. The Saimiri brain endothelial cell clones (SBEC) varied in their expression of different surface molecules. For example, various combinations of receptors involved in P. falciparum PRBC adherence such as CD36, ICAM-1 and E-selectin, were expressed at baseline values and could be up-regulated by human srTNF-alpha and human srIFN-gamma. One of the SBEC clones showed a strong cytoadherence for various laboratory strains of P. falciparum despite the absence of surface expression of any of the known endothelial receptors implicated in PRBC adherence. This finding suggests the existence of a new and uncharacterized PRBC binding receptor. The use of target organ specific endothelial cell lines expressing a number of different potential P. falciparum PRBC cytoadherence receptors, will be a useful in vitro system for the evaluation of strategies for the development of vaccine and antimalarial drugs to prevent human cerebral malaria.
Pathogenic mechanisms in human cerebral malaria remain unclear. We reevaluate the role of cell-mediated immune mechanisms in the pathogenesis of this disease based on autopsy findings in a 34-year-old Caucasian male. Histologic examination of brain tissue showed typical features of severe malaria infection (sequestration of Plasmodium falciparum-infected erythrocytes in vessels, cerebral oedema, petechial lesions and Dürck granulomas). In addition to these classical changes, we found that leukocytes that stained positively in immunohistochemistry for CD68 and tumor necrosis factor-alpha (TNF) coexisted with infected erythrocytes in capillaries, whereas in venules the monocyte population outnumbered the erythrocytes. Notable expression of ICAM-1 on endothelial cell surface was detected by immunohistochemistry in vessels with sequestered cells but not in unaffected vessels. These changes are identical to those of the murine model of the disease, in which cell-mediated immune mechanisms and TNF have been implicated. In vitro, ICAM-1 has been shown to be a potential ligand for P. falciparum-infected erythrocytes. In malaria patients, high serum TNF levels, which have been detected in close correlation with disease severity, may thus favor adhesion to endothelial cells of either red or white blood cells via enhanced ICAM-1 expression. The present observations are further evidence for a role of cell-mediated immunity in the pathogenesis of human cerebral malaria.
Recirculating lymphocytes migrate into areas of lung inflammation by binding to microvascular endothelium and transmigrating into extravascular tissue. In this report, we examined the multiple-step paradigm using a unique system: recirculating lymphocytes from sheep peripheral lymphatics adhering to activated lung microvascular endothelium in conditions of physiologic flow. Video microscopy demonstrated that recirculating lymphocytes formed abrupt adhesions, without requisite rolling, on the lung microvascular endothelial cells. Lymphocyte velocity was unchanged within 100 ms of the development of firm adhesions. To dissect the adhesion mechanism, the lymphocytes were pretreated with anti-LFA-1 or anti-L-selectin monoclonal antibody (mAb). Both mAb decreased the incidence of firm adhesions. The mechanism of this inhibition was investigated using time-lapse topographic reconstructions of cell movement after pretreatment with mAb. Time-lapse analysis of the movement of lymphocytes pretreated with anti-LFA-1 mAb suggested that abortive adhesion was manifested by a characteristic saltatory movement and a sustained reduction in cell velocity (rolling) to <25 microns/s. In contrast, abortive adhesions of lymphocytes pretreated with anti-L-selectin mAb demonstrated transient arrest (tethering) but minimal rolling before resumption of baseline velocity in the flow stream. These observations provide insights into selectin and integrin regulation of lymphocyte transmigration into the lung. Further, the results of mAb inhibition suggest that the mechanism of lymphocyte migration may have some unique features not observed in studies of neutrophil transmigration.
The purpose of this study was to assess the phenotypic and functional characteristics of pulmonary microvascular endothelial cells (MVEC) in the acute respiratory distress syndrome (ARDS). Pulmonary MVEC were isolated from the lungs of five patients who developed ARDS, and from four patients who had undergone a lobectomy for lung carcinoma, as controls. Adhesion molecules and other surface molecules were quantitated on these cells by flow cytometry and the cytokines IL-6 and IL-8 were measured in the supernatants by ELISA. The constitutive expression of intercellular adhesion molecule and, to a lesser extent, vascular adhesion molecule-1, was significantly increased on MVEC isolated from all ARDS patients, as compared with control MVEC. CD14 and TNF receptor p75 were also increased on the surface of MVEC isolated from most patients with ARDS. The expression of ELAM-1 and TNF receptor p55 (TNF-R1) was not significant on the surface of either ARDS-derived or control pulmonary MVEC. The constitutive ability of ARDS-derived MVEC to secrete IL-6 and IL-8 was markedly enhanced as compared with control MVEC. Upon in vitro restimulation by TNF, pulmonary MVEC from ARDS patients showed lower ICAM-1 upregulation, but similar IL-6 and IL-8 production capacity, when compared with control MVEC. Selective differences were found in cell adhesion molecules and TNF receptor p75 expression on pulmonary MVEC isolated from patients with ARDS. These pulmonary MVEC spontaneously overexpress some adhesion molecules and produce greater amounts of the pro- and anti-inflammatory cytokines IL-8 and IL-6. These findings suggest that ICAM-1 and TNF receptor p75 may have a particular involvement in the pathogenesis of acute lung injury, and that the endothelium may be an important source of cytokines detected in broncho-alveolar lavage during this syndrome. It is tempting to hypothesize that the differences observed result from either a genetic predisposition to ARDS based on MVEC phenotype or to a long-lived MVEC phenotypic change induced by ARDS. By allowing the monitoring of phenotypic and functional parameters, cultures of pulmonary MVEC isolated from ARDS patients may thus represent a useful system to analyze further the mechanisms of acute lung injury and to evaluate the efficacy of drugs, including inhibitors of cytokines and of adhesion molecules.
Women are particularly susceptible to malaria during first and second pregnancies, even though they may have developed immunity over years of residence in endemic areas. Plasmodium falciparum-infected red blood cells (IRBCs) were obtained from human placentas. These IRBCs bound to purified chondroitin sulfate A (CSA) but not to other extracellular matrix proteins or to other known IRBC receptors. IRBCs from nonpregnant donors did not bind to CSA. Placental IRBCs adhered to sections of fresh-frozen human placenta with an anatomic distribution similar to that of naturally infected placentas, and this adhesion was competitively inhibited by purified CSA. Thus, adhesion to CSA appears to select for a subpopulation of parasites that causes maternal malaria.
Adherence of Plasmodium falciparum parasitized erythrocytes to the microvascular endothelium is mediated by different receptors expressed by endothelial cells. The study of the adherence of P. falciparum-infected erythrocytes to Saimiri monkey brain microvascular endothelial cells revealed the presence of an additional receptor, which was identified and further characterized. This receptor was also found on the surface of primary human lung endothelial cells (HLEC). We developed two mAbs to this receptor which very efficiently blocked the adherence of parasite strains to Saimiri brain endothelial cells (SBEC). The ability of these mAb to bind to SBEC was partially blocked by chondroitin-4-sulphate (CSA). Competitive inhibition assays on adherence of parasitized red blood cells (PRBC) showed that CSA, but not hyaluronic acid, chondroitin-6-sulphate, dermatan sulphate, keratane sulphate, heparan sulphate or chondroitin-4S-disaccharide, was able to almost completely inhibit PRBC adherence. The same effect was obtained with chondroitinase ABC and AC, but not B, hyaluronidase or heparinase. These results strongly suggest that a member of the chondroitin-glycosaminoglycan family, CSA, represents an additional receptor used by P. falciparum PRBC to cytoadhere to microvascular endothelial cells.
Plasmodium falciparum-infected erythrocytes (IRBC) roll on the adhesion molecule P-selectin in vitro under flow conditions that approximate the shear stress in capillary and postcapillary venules in which cytoadherence occurs in vivo. The pathological significance of this adhesive interaction is currently unknown. In this study, we further investigated the molecular interactions between IRBC and P-selectin by using a laminar flow system that allowed for the direct visualization of IRBC-substratum interactions. The results showed that the IRBC-P-selectin interaction was Ca2+-dependent and involved the lectin domain of P-selectin and a sialic acid residue on IRBC. The sialylated P-selectin ligand was trypsin-sensitive, which suggests that it could be part of the parasite antigen PfEMP1 that interacts with CD36 and intercellular adhesion molecule-1 (ICAM-1), but different from a trypsin-resistant IRBC ligand that adheres selectively to chondroitin sulfate A. Studies on the rolling and adhesion of IRBC on activated platelets that express both CD36 and P-selectin showed that inhibition of rolling on P-selectin reduced the adhesion of some clinical parasite isolates to CD36, whereas other parasite isolates appeared to interact directly with CD36. Thus, cytoadherence under physiological flow conditions may be mediated by multiple IRBC ligands that interact with different adhesion molecules in a cooperative fashion. These findings underscore the complexity of the interactions betweeen IRBC and vascular endothelium.
Some complications of Plasmodium falciparum infection such as cerebral malaria and pregnancy-associated malaria may be partially due to cytoadherence of erythrocytes infected by mature parasites on microvascular endothelial cells or placental syncytiotrophoblasts. Recently a new cytoadherence receptor, chondroitin-4-sulphate (CSA), was identified first on endothelial cells in primates and then on CHO cells and purified receptors. Further study has implicated CSA in cytoadherence of infected red blood cells to syncytiotrophoblasts in human placenta and Saimiri sciureus monkeys. In solution the minimal size for full inhibitory effect is approximately 9 kDa. Injection of CSA in Plasmodium falciparum-infected Saimiri monkeys resulted in specific release of sequestered erythrocytes infected by mature parasites. An added interest of these findings is that CSA, a glycosaminoglycan, is already in clinical use for treatment of degenerative joint disease. Current data on the parasite ligand for CSA indicates that it is not co-expressed with other cytoadherence ligands and that its binding activity decreases as the parasite matures from the 20th to 40th hour of the cycle. Since one or more var genes encoding the CSA ligand have been identified, it is likely that peptides will be obtained quickly and used either for direct inhibition of cytoadherence on CSA or for development of an anti-sequestration vaccine.
Microbial pathogens subvert host adhesion molecules to disseminate or to enter host cells to promote their own survival. One such subversion is the cytoadherence of Plasmodium falciparum-infected erythrocytes (IRBC) to vascular endothelium, which protects the parasite from being removed by the spleen. The process results in microcirculatory obstruction and subsequent hypoxia, metabolic disturbances, and multiorgan failure, which are detrimental to the host. Understanding the molecular events involved in these adhesive interactions is therefore critical both in terms of pathogenesis and implications for therapeutic intervention. Under physiological flow conditions, cytoadherence occurs in a stepwise fashion through parasite ligands expressed on the surface of IRBC and the endothelial receptors CD36, intercellular adhesion molecule-1 (ICAM-1), P-selectin, and vascular adhesion molecule-1. Moreover, rolling on ICAM-1 and P-selectin increases subsequent adhesion to CD36, indicating that receptors can act synergistically. Cytoadherence may activate intracellular signaling pathways in both endothelial cells and IRBC, leading to gene expression of mediators such as cytokines, which could modify the outcome of the infection.
Microvascular sequestration was assessed in the brains of 50 Thai and Vietnamese patients who died from severe malaria (Plasmodium falciparum, 49; P. vivax, 1). Malaria parasites were sequestered in 46 cases; in 3 intravascular malaria pigment but no parasites were evident; and in the P. vivax case there was no sequestration. Cerebrovascular endothelial expression of the putative cytoadherence receptors ICAM-1, VCAM-1, E-selectin, and chondroitin sulfate and also HLA class II was increased. The median (range) ratio of cerebral to peripheral blood parasitemia was 40 (1.8 to 1500). Within the same brain different vessels had discrete but different populations of parasites, indicating that the adhesion characteristics of cerebrovascular endothelium change asynchronously during malaria and also that significant recirculation of parasitized erythrocytes following sequestration is unlikely. The median (range) ratio of schizonts to trophozoites (0.15:1; 0.0 to 11.7) was significantly lower than predicted from the parasite life cycle (P < 0.001). Antimalarial treatment arrests development at the trophozoite stages which remain sequestered in the brain. There were significantly more ring form parasites (age < 26 hours) in the cerebral microvasculature (median range: 19%; 0-90%) than expected from free mixing of these cells in the systemic circulation (median range ring parasitemia: 1.8%; 0-36.2%). All developmental stages of P. falciparum are sequestered in the brain in severe malaria.
Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to chondroitin-4-sulphate (CSA) is inhibited by soluble CSA in vitro on Saimiri brain microvascular endothelial cells (SBEC) and in vivo in P. falciparum-infected Saimiri monkeys. We tested whether the SBEC model was appropriate for studying CSA-binding IRBC using four cell lines. All SBEC expressed a chondroitin sulphate (CS), with a composition of CSA. The mean sizes of these CSA were 20.5, 22, 23, 32.5 and 36 kDa for SBEC 3A and C2, CHO, SBEC 1D and 17, respectively. We found that cytoadhesion of the Palo-Alto (FUP)1 CSA-binding phenotype, selected by panning on SBEC 17, was specifically inhibited in a dose-dependent manner by all the purified CSA. The extent of inhibition depended on the cellular origin of the tested CSA. SBEC 17 CSA was 33 times more efficient than CHO-CSA and 21 times more efficient than the 50 kDa commercial bovine trachaea CSA. Dynabeads coated with a total extract of SBEC 1D CS-proteoglycans interacted with CSA- but not with CD36- or ICAM-1-binding IRBC. These Dynabeads also interacted specifically with the PfEMP1 DBL-3 domain, on the surface of CHO transfectants, but not with the CIDR-1 domain. Thrombomodulin was involved in IRBC adhesion to all SBEC whereas CD44 was only expressed by SBEC 1D and 17. These two CSA-proteoglycans have also been detected at the surface of human endothelial cells. Thus, the two homologous models, SBEC/Saimiri sciureus, are useful and reliable tools for the evaluation of new anti-CSA adhesion treatments and anti-disease vaccines for pregnant women.
Falciparum malaria affect all ages with multiple-systemic complications which varies in different age group. We studied 242 children with complicated Falciparum malaria with a median age of 6.5 years to look for occurrence of different complications in younger and older age groups and overall mortality picture. Unarousable coma (40.5%), severe anemia (26.03%), repeated seizures (46.2%) and hepatopathy (32.2%) were commonest complications. Under five children had higher risk of development of cerebral malaria (P<0.01), severe anemia (P<0.05) and seizures (P<0.001); whereas above five children had higher risk of acute renal failure (P<0.05) and malarial hepatopathy (P<0.02). Over all mortality was 9.9%, cerebral malaria being the commonest cause (6.6%). Multi-system involvement was seen in 58.4% cases of death. Children having pulmonary edema, shock and cerebral malaria had high case fatality rate.
Fibrin thrombi in the brain in fatal pediatric malaria correlate with malarial pigment globules
- P B Chandak
- R A Carr
- P T Seed
Chandak PB, Carr RA, Seed PT, et al. Fibrin thrombi in the brain in
fatal pediatric malaria correlate with malarial pigment globules [abstract 297]. In: Programs and abstracts of the 48th meeting of the
American Society of Tropical Medicine and Hygiene (Washington, DC).
Ex vivo deseques-tration of Plasmodiumfalciparum–infectederythrocytesfromhumanpla-centa by chondroitin sulfate A
- J Gysin
- B Pouvelle
- N Fievet
- A Scherf
- Lepolard
Gysin J, Pouvelle B, Fievet N, Scherf A, Lepolard C. Ex vivo deseques-tration of Plasmodiumfalciparum–infectederythrocytesfromhumanpla-centa by chondroitin sulfate A. Infect Immun 1999;67:6596–602
Severe falciparum malaria
- PF Beales
- B Brabin
- E Dorman