Malaria is an important neglected disease and one of the most important global health problems, potentially affecting more than one third of the world's population. Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, associated with a 10–14% mortality rate and approximately 1–2 million annual deaths among young children predominantly in sub-Saharan Africa and Southeast Asia, yet its pathogenesis remains incompletely understood. In Ghana, malaria has a wide spectrum of presentations: from asymptomatic carriers to mild malaria to multifactorial severe disease, including CM and severe malarial anemia (SMA) [1–5].
CM, a clinically complex syndrome of coma and potentially reversible encephalopathy, is associated with increased levels of proinflammatory cytokines like tumor necrosis factor (TNF)-α, interferon (IFN)-γ and lymphotoxin [6, 7], and increasingly recognized long-term sequelae in survivors [7–15]. Although the physiopathology of CM has been extensively investigated, the exact cellular and molecular basis of the neuropathology is still unclear. Recent studies have shown that mechanical blockage caused by sequestration of parasitized red blood cells (pRBCs), leukocytes and platelets [3, 4, 8–12], secretion of cytokines and chemokines [2, 6, 7, 13], angiogenic failure [14, 15], immune status and the genetic background of the host, and parasite factors [7, 16] are involved in the pathogenesis of CM. However, it is generally accepted that two major factors are involved: (i) metabolic insufficiencies due to the sequestration of pRBCs, leukocytes and platelets within brain vessels via upregulated adhesion molecules [3, 4, 8–12], and (ii) immunological reactions with the local involvement of T cells and monocytes activated by Plasmodium antigens [7, 11]. These two major mechanisms appear to act together under the control of cytokines , and chemokines  to exacerbate CM.
Postmortem data in humans and murine models of CM show that neuronal damage in brain tissue occurs in CM, although the parasites remain confined to the intravascular space (with no contact with neurons). This strongly suggests that the blood-brain barrier (BBB) is perturbed, and thus the BBB represents a key interface between the intraerythrocytic stages of the parasite and the human host. The functional and morphological evidence supports mild-to-moderate impairment of the BBB, but whether this is sufficient to cause neurological complications such as CM is inconclusive [16, 17]. Mechanical blockage could occur from the ability of pRBC's to adhere to unparasitized erythrocytes and endothelial cells, and sequester in the deep cerebral microvasculature [3, 4, 8–12]. Parasite sequestration alone, however, cannot account for fatal CM pathogenesis as there is evidence that survivors of CM have the same degree of sequestration during comas as those succumbing to disease. It is evident that host immune factors play an important role in the pathogenesis of CM. [8, 16]. Although parasite sequestration during severe malaria is central to pathogenesis of severe malaria, the role of cytokines, chemokines, apoptotic, and angiogenic factors in exacerbating disease severity remains unclear.
The balance between specific cytokines and chemokines produced in response to infection with Plasmodium falciparum is thought to play an important role in CM and other forms of severe malaria. Severe malaria has been associated with high TNF-α plasma levels in conjunction with increased production of IFN-γ and IL-1β [18, 19] and decreased production of anti-inflammatory cytokines, notably IL-10 and TGF-β [1, 19–22]. Pro-inflammatory Th1-type cytokines (eg. TNF-α, IFN-γ, interleukin IL-1β, and IL-6) are thought to be critical to the control of exoerythrocytic and erythrocytic Plasmodium falciparum infection [23, 24], but their exaggerated production may also contribute to organ damage, particularly in the brain. It is widely accepted that anti-inflammatory Th2-type cytokines down-regulate Th1-derived cytokines. Th2-type cytokines, such as IL-10, has been shown to regulate Th1-cytokines and prevent CM in some animal models . The regulation of TNF-α levels by IL-10 appears to contribute to the prevention of severe malarial anemia in humans [1, 20, 21]. However, the role that IL-10 plays may depend on its levels, since very high levels of IL-10 have been associated with severe malaria in humans  and some animal models . Therefore, cytokines appear to maintain a delicate balance between the control of infection and contribution to disease in falciparum malaria infection. However, the expression of Th1 and Th2 cytokines in CSF, either from the peripheral circulation via the BBB or from neuronal immune cells (glia) has not been adequately addressed.
Chemokines, or chemoattractant cytokines, and their corresponding receptors have also been shown to mediate mobilization and coordination of immune responses to malaria. Chemokines have lympho-chemotactic activity and modulate many infectious and inflammatory diseases, including malaria. The recent demonstration of leukocyte sequestration, in addition to pRBC sequestration [3, 4, 8–12], within brain vessels in human CM suggests a more important role for leukocytes, including eosinophils, in CM immunopathology than previously thought. Thus, chemokines, including eotaxin, may play an important role in human CM by attracting leukocytes to sequestration sites. Chemokines are less well studied in severe malaria, but recent studies have associated severe malaria infection with increased production of chemokines of the C-C or β subfamily, including regulated upon activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and IL-8 [2, 13, 26–28]. MCP-1, MIP-1α and MIP-1β are potent chemoattractants for monocytes to produce TNF-α and IL-6. IL-8 preferentially recruits neutrophils and plays an important role in inflammatory diseases. Recently, low levels of RANTES have been associated with severe malaria [26, 27, 29], and specifically associated with mortality in children with CM . The low levels of RANTES in severe malaria have been associated with malaria-induced thrombocytopenia [26, 29], given that platelets are a major reservoir of RANTES in the peripheral circulation. In contrast, increased mRNA and protein expression of RANTES and CCR5 was found in localized brain regions of children dying of CM . Interferon inducible protein 10 (IP-10) is a member of the CXC or α subfamily of chemokines, and is induced in response to IFN-γ attracting activated Th1 cells . IP-10 levels have been shown to increase in cultured intervillous blood mononuclear cells isolated from placenta's infected with malaria [31, 32], although no studies have characterized IP-10 levels in human cerebral malaria. Hanum and colleagues recently demonstrated the induction of IP-10 expression in the brain of both CM-susceptible (C57BL/6) and CM-resistant (BALB/c) mice as early as 24 hours post-infection with Plasmodium berghei ANKA, and in KT-5 astrocyte cell line in vitro upon stimulation with a crude antigen of malaria parasites . Currently, the role of chemokines in clinical severity and outcome of malaria, especially the development of CM and SMA in children remains poorly defined.
Angiogenic factors, long implicated as prognostic factors in cerebral ischemia or stroke , have been suggested to play a role in the petechial hemorrhages and BBB dysfunction associated with CM pathology [14, 15]. Vascular endothelial growth factor (VEGF) stimulates endothelial cell growth and migration as well as enhancing vascular permeability. VEGF levels (more VEGF+ astrocytes) were higher in CM patients as compared with controls in a post-mortem immunohistology study of CM patients . Platelets, that accumulate with pRBCs in the brain miscrovasculature in CM patients [10, 12], are also implicated in CM pathology through TGF-β induced apoptosis in TNF activated human brain endothelial cells [35, 36]. Platelet derived growth factor (PDGF) is another angiogenic factor that stimulates vascular growth, and has been implicated as a neuroprotective factor inducing regeneration of damaged axons and neuronal growth after ischemia . These angiogenic factors play a dominant role in the recovery from stroke and may be applicable in CM due to their effect on the endothelium, which is central to CM pathology. These angiogenic factors most probably impact the regenerative potential of the parasite-induced BBB damage, rather than impacting neoangiogenesis, since CM is an acute neurological syndrome.
Parasite-induced apoptosis in the host may also mediate the severity of malaria. High levels of Fas-Ligand in sera of human [38, 39], monkey , and mice  are associated with severity of malaria. Lymphocytes and macrophages express increased levels of Fas and Fas-Ligand during an acute Plasmodium chabaudi infection . TNFR2-deficient mice are resistant to experimental cerebral malaria (ECM), Fas-deficient mice showed 50% reduction in ECM incidence, and TNFR1-deficient mice showed the least reduction in ECM incidence [42–46]. Lpr & Gld mice, deficient in Fas & Fas-Ligand, are protected from fatal ECM . The presence of these apoptotic factors in CSF and serum, and their relevance in CM and CM-associated mortality has not been fully investigated. The rapid reversibility of the clinical symptoms of CM suggests that tissue necrosis is unlikely to occur [9, 16, 48], making apoptosis a more likely pathogenic mechanism.
Recently, new strategies including magnetic resonance imaging and ophthalmological evaluation of children with CM have been proposed as clinically useful predictors of CM severity, but their reliability is being evaluated. The study hypothesis was that parasite-induced dysregulation in the levels of inflammatory, apoptotic and angiogenic factors at the time of CM death would predict mortality risk of CM. The goal of this study was to identify factors that are tightly associated with CM mortality in Ghanaian children for further development as biomarkers of CM disease. The present study employed a high throughput multiplexed immunoassay to evaluate the predictive value of serum and CSF levels of key immunomodulators (inflammatory, apoptotic and angiogenic proteins) in determining mortality risk in severe malaria in Ghanaian children. We investigated the serum and CSF profiles of 36 different biomarkers (IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGF basic protein, CRP, G-CSF, GM-CSF, IFN-γ, TNF-α, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, RANTES, SDF-1α, CXCL11 (I-TAC), Fas-ligand [Fas-L], soluble Fas [sFas], sTNF-R1 (p55), sTNF-R2 (p75), MMP-9, TGF-β1, PDGF bb and VEGF) in order to identify the immune factors which influence progression to fatal outcomes associated with CM.