Cerebral malaria is a major life-threatening complication of Plasmodium falciparum infection in humans. However, the mechanisms underlying fatal cerebral complications are still not fully understood. The present study was conducted to determine immunologically-relevant biological factors known to be associated with fatal CM and other severe forms of malaria in India. We examined disease associated inflammatory mediators, including cytokines, chemokines, apoptotic and angiogenic factors in sera of patients with HC, MM, CMS and CMNS. Due to the lack of concurrent studies of inflammatory, apoptotic and angiogenic biomarkers in appropriate time-matched controls from other diseases, the results are discussed in context. The study was conducted in an area of P. falciparum transmission where life-threatening complications of malaria occur.
The involvement of sTNF-R, sFas and IL-10 in the pathogenesis of CM and its associated mortality is consistent with previous reports. Mice deficient in TNF-R2 (TNF-R2-/-) or Fas ligand (Fas-/-) survive longer than wild type mice in experimental studies of CM, while TNF-R2-/- mice survive the longest in the absence of anti-malarial treatment . The critical role of TNF-R2 in CM-associated death in experimental malaria has been well established [39–41]. In the absence of TNF-R2, mortality was eliminated in mice , while the restoration of TNF-R2 expression in brain tissue induced lethal CM . Elevated levels of sTNF-R1 and sTNF-R2 in African children afflicted with CM have been reported, but no significant correlation with CM-associated mortality has been demonstrated [40, 42]. This study has shown that plasma levels of sTNF-R2 are significantly higher in CMNS than in other groups. Since epidemiological and phenotypic differences exist between Indian and West African CM, further studies on the validity of these findings in South American, and other Asian settings may clarify these differences.
sFas and its ligand, Fas-L, are critical factors mediating malaria pathogenesis in both murine CM models and in human CM. Fas-deficient mice are protected against CM, while Fas and Fas-L expression increased in brain tissue of wild type mice with CM . Fas and Fas-L interactions activate the classical extrinsic apoptotic pathway involving caspase cascades (caspase-8, -9, and -3) and activated caspase-3-positive apoptotic astrocytes have been found in mice affected by CM . In a recent study in Gabon, Touré et al  demonstrated that although the factors inducing apoptosis have not been identified, apoptosis may play an important role in triggering some of the neurological manifestation associated with severe malaria. The present study has confirmed a robust positive correlation between sFas levels and CM fatality as was observed in postmortem CSF and serum in Ghanaian CM patients . Thus, efforts are underway to test postmortem tissues and CSF from this Indian population in future investigations relating to CM mediated apoptosis and severity.
Mean IL-10 levels increased in the CMS group in comparison to the HC group but the levels declined in the CMNS group when compared to CMS. Interestingly, this decline coincided with a significant increase in IP-10 production as previously reported . Interleukin-10 (IL-10) is a pleiotropic cytokine that inhibits the production of interferon-gamma (IFN-γ) by T helper 1 (Thl) cells and TNF secretion by macrophages. The protective role of IL-10 in CM severity has previously been demonstrated in murine CM models. In vivo neutralization of endogenous IL-10 in CM-resistant mice resulted in CM while increased production of IL-10 inhibited P. berghei ANKA antigen-specific IFN-γ and protected against CM in susceptible mice . Recent studies have shown elevated levels of circulating IL-10 in patients with cerebral and severe malaria but less so in mild malaria. Thus, it seems that the decreased production of IL-10 in non survivors (CMNS) probably enhances production of IFN-γ and IP-10; an interferon inducible protein, to increase CM severity. Further mechanistic study is needed to determine the role of IL-10 in fatal outcomes of CM.
The study also revealed that VEGF may play a protective role against mortality associated with CM. VEGF is a vascular permeability factor with potent angiogenic and wound healing properties . VEGF also interacts with PDGFbb and other factors such as thrombospondin-1 (TSP-1) and transforming growth factor beta (TGF-β) . There is substantial evidence demonstrating the importance of VEGF in brain tissue repair and in CNS wound healing [48, 49]. Exogenous administration of VEGF to injured brain tissues improved wound repair, functional response and decreased apoptosis in rats . Decreased levels of serum VEGF and PDGFbb correlated with fatal outcome in CM. Interestingly, a decrease in levels of PDGFbb has been reported in CSF and serum of non survivors of CM . Whether the decline in levels of VEGF or PDGFbb are causative agents of CM mortality or a specific phenotype of patients with susceptibility to fatal CM needs further evaluation. Based on this observation, it seems that significant decreases in VEGF may exacerbate neurological damage triggered by inflammatory- and/or parasite-derived factors, thus leading to death. It has been shown that neutralization of endogenous VEGF in the brains of injured rats results in decreased angiogenic activity and increased vascular and astrocyte degeneration, leading to larger wound cavities . It is unclear why or how VEGF and PDGFbb production declines in the peripheral blood of CM patients. VEGF expression is negligible in the CNS under normal conditions, and it is activated after various types of neurological injuries . In addition, elevated levels of VEGF-positive astrocytes and deposition of VEGF receptor-1 (VEGFR-1 or Flt-1) in Durck's granulomas were found in non-surviving CM patients . Recently, elevated levels of VEGF in P. falciparum -infected primigravid mothers were reported . Thus, it is evident that VEGF is involved in severe manifestations of malaria, although further studies will be required to fully understand its role in fatal outcomes of severe malaria. In a recent study involving Kenyan children, it was demonstrated that erythropoietin but not VEGF was associated with neurological sequelae . It remains to be determined whether the differences between our study findings and the Kenyan study is due to differences in malaria epidemiology, immune status of the patients or host population genetics.
IP-10 (CXCL10) is a chemokine that is induced by IFN-γ, TNF-α, and other factors and has chemotactic activity for activated Th1 lymphocytes. There is growing evidence implicating this chemokine in both infectious and noninfectious causes of neuronal injury, dementia and inhibition of angiogenesis [52–54]. In this study, IP-10 levels were remarkably elevated as disease severity increases, with highest levels among non-survivors of CM (CMNS). These findings are consistent with recent studies on sera obtained from CM non-survivors in Ghana  as well as other reports implicating inflammatory and apoptotic mechanisms in the pathogenesis of CM [8–12].
This study confirmed an observed link between elevated levels of transcripts of genes implicating interferon-regulated processes and apoptosis in a murine CM model  and of elevated IP-10 in CM patients with poor prognosis . It seems that TNF-α which is activated following the release of malaria antigens after schizont rupture, may activate production of IP-10 in brain capillaries and astrocytes [56, 57]. IP-10 in concert with TNF-α can cause vascular injury resulting in breakdown in the blood brain barrier, which leads to accumulation of leukocytes that induce local hyper-inflammation. Significant down regulation of VEGF may further inhibit angiogenesis and regeneration of damaged blood capillaries. High systemic levels of IP-10, with its strong angiostatic properties, in CM patients may further inhibit angiogenesis and interfere with VEGF function. IP-10 also blocks VEGF interaction with its natural ligand by interacting with cell-surface glycosaminoglycans which provide accessory support for VEGF . In viral meningitis, high levels of IP-10 in the cerebrospinal fluid (CSF) have been reported . High levels of IP-10 have also been found in the CSF of patients with HIV-associated dementia correlated with neuropsychiatric impairment . Furthermore, mouse studies have demonstrated that the HIV-1 virus-encoded protein gp120 directly activates astrocytes to produce IP-10 using a novel mechanism independent of IFN-α and the STAT-1 pathway of IP-10 induction . In addition, elevated levels of IP-10, especially from astrcoytes, have been detected in Japanese encephalitis patients . Based on these findings and those from other infectious disease models, it appears that IP-10 may play a more important role in CM-related brain encephalopathy/injury than previously thought. A very recent experimental CM study using knockout mice which lacked either IP-10, Mig or their receptor CXCR3, demonstrated that these molecules played a critical role in the CM associated death caused by P. berghei ANKA infection. It was further shown that IP-10 and Mig play a role in attracting CD8+T cells and NK cells to brains of infected mice and may be involved in causing death. In the infected mice, brain endothelial cells lining blood capillaries expressed both IP-10 and Mig while the neurons expressed IP-10 . Collectively, these evidences suggest a prominent role for IP-10 in the pathogenesis of CM and associated death.
The inverse relationship between inflammatory factors and apoptotic and angiogenic markers were striking. In previous studies, the IL-10 (anti-inflammatory) to TNF-α (pro-inflammatory) ratio had been shown to be lower in severe malaria anaemia than in controls . Interestingly, in this study sTNF-R2/VEGF, IP-10/VEGF and sFas/VEGF ratios were highest in the CMNS group; and were at least 10-fold higher as compared to the CMS group. This difference supports the hypothesis that the relative balance in inflammatory markers and apoptotic or angiogenic factors, especially VEGF, may predict risk of CM-associated mortality. These ratios could be used as prognostic markers in predicting fatal or severe outcomes associated with CM.