Sequestration and microvascular congestion are associated with coma in human cerebral malaria.

The pathogenesis of coma in severe Plasmodium falciparum malaria remains poorly understood. Obstruction of the brain microvasculature because of sequestration of parasitized red blood cells (pRBCs) represents one mechanism that could contribute to coma in cerebral malaria. Quantitative postmortem microscopy of brain sections from Vietnamese adults dying of malaria confirmed that sequestration in the cerebral microvasculature was significantly higher in patients with cerebral malaria (CM; n = 21) than in patients with non-CM (n = 23). Sequestration of pRBCs and CM was also significantly associated with increased microvascular congestion by infected and uninfected erythrocytes. Clinicopathological correlation showed that sequestration and congestion were significantly associated with deeper levels of premortem coma and shorter time to death. Microvascular congestion and sequestration were highly correlated as microscopic findings but were independent predictors of a clinical diagnosis of CM. Increased microvascular congestion accompanies coma in CM, associated with parasite sequestration in the cerebral microvasculature.

Coma in fatal adult human malaria is not caused by cerebral oedema.

BACKGROUND: The role of brain oedema in the pathophysiology of cerebral malaria is controversial. Coma associated with severe Plasmodium falciparum malaria is multifactorial, but associated with histological evidence of parasitized erythrocyte sequestration and resultant microvascular congestion in cerebral vessels. To determine whether these changes cause breakdown of the blood-brain barrier and resultant perivascular or parenchymal cerebral oedema, histology, immunohistochemistry and image analysis were used to define the prevalence of histological patterns of oedema and the expression of specific molecular pathways involved in water balance in the brain in adults with fatal falciparum malaria. METHODS: The brains of 20 adult Vietnamese patients who died of severe malaria were examined for evidence of disrupted vascular integrity. Immunohistochemistry and image analysis was performed on brainstem sections for activation of the vascular endothelial growth factor (VEGF) receptor 2 and expression of the aquaporin 4 (AQP4) water channel protein. Fibrinogen immunostaining was assessed as evidence of blood-brain barrier leakage and perivascular oedema formation. Correlations were performed with clinical, biochemical and neuropathological parameters of severe malaria infection. RESULTS: The presence of oedema, plasma protein leakage and evidence of VEGF signalling were heterogeneous in fatal falciparum malaria and did not correlate with pre-mortem coma. Differences in vascular integrity were observed between brain regions with the greatest prevalence of disruption in the brainstem, compared to the cortex or midbrain. There was a statistically non-significant trend towards higher AQP4 staining in the brainstem of cases that presented with coma (P = .02). CONCLUSIONS: Histological evidence of cerebral oedema or immunohistochemical evidence of localised loss of vascular integrity did not correlate with the occurrence of pre-mortem coma in adults with fatal falciparum malaria. Enhanced expression of AQP4 water channels in the brainstem may, therefore, reflect a mix of both neuropathological or attempted neuroprotective responses to oedema formation.

Induction of the vascular endothelial growth factor pathway in the brain of adults with fatal falciparum malaria is a non-specific response to severe disease.

AIMS: Pathological or neuroprotective mechanisms in the brain in severe malaria may arise from microvascular obstruction with malaria-parasitized erythrocytes. This study aimed to investigate the role of hypoxia and induction of the vascular endothelial growth factor (VEGF) pathway in the neuropathophysiology of severe malaria. METHODS AND RESULTS: Immunohistochemistry was performed on post mortem brain tissue sections from 20 cases of severe malaria and examined for the expression of transcriptional regulators of VEGF [hypoxia-inducible factor-1 alpha (HIF-1alpha), HIF-2alpha], DEC-1, VEGF, VEGF receptors 1 and 2, and the activated, phosphorylated VEGF receptor 2 (pKDR). HIFs showed limited protein expression and/or translocation to cell nuclei in severe malaria, but DEC-1, which is more stable and regulated by HIF-1alpha, was observed. There was heterogeneous expression of VEGF and its receptors in severe malaria and non-malarial disease controls. pKDR expression on vessels was greater in malaria cases than in controls but did not correlate with parasite sequestration. VEGF uptake by malaria parasites was observed. CONCLUSIONS: VEGF and its receptor expression levels in severe malaria reflect a non-specific response to severe systemic disease. Potential manipulation of events at the vasculature by the parasite requires further investigation.

Erythropoietin and its receptors in the brainstem of adults with fatal falciparum malaria.

BACKGROUND: Facilitation of endogenous neuroprotective pathways, such as the erythropoietin (Epo) pathway, has been proposed as adjuvant treatment strategies in cerebral malaria. Whether different endogenous protein expression levels of Epo or differences in the abundance of its receptor components could account for the extent of structural neuropathological changes or neurological complications in adults with severe malaria was investigated. METHODS: High sensitivity immunohistochemistry was used to assess the frequency, distribution and concordance of Epo and components of its homodimeric and heteromeric receptors, Epo receptor and CD131, within the brainstem of adults who died of severe malaria. The following relationships with Epo and its receptor components were also defined: (i) sequestration and indicators of hypoxia; (ii) vascular damage in the form of plasma protein leakage and haemorrhage; (iii) clinical complications and neuropathological features of severe malaria disease. Brainstems of patients dying in the UK from unrelated non-infectious causes were examined for comparison. RESULTS: The incidence of endogenous Epo in parenchymal brain cells did not greatly differ between severe malaria and non-neurological UK controls at the time of death. However, EpoR and CD131 labelling of neurons was greater in severe malaria compared with non-neurological controls (P = .009). EpoR labelling of vessels was positively correlated with admission peripheral parasite count (P = .01) and cerebral sequestration (P < .0001). There was a strong negative correlation between arterial oxygen saturation and EpoR labelling of glia (P = .001). There were no significant correlations with indicators of vascular damage, neuronal chromatolysis, axonal swelling or vital organ failure. CONCLUSION: Cells within the brainstem of severe malaria patients showed protein expression of Epo and its receptor components. However, the incidence of endogeneous expression did not reflect protection from vascular or neuronal injury, and/or clinical manifestations, such as coma. These findings do not provide support for Epo as an adjuvant neuroprotective agent in adults with severe malaria.

Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells.

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.

Axonal and astrocyte injury markers in the cerebrospinal fluid of Kenyan children with severe malaria.

A retrospective study of cerebrospinal fluid (CSF) levels of markers of brain parenchymal damage was conducted in Kenyan children with severe falciparum malaria. Two markers were analysed by immunoassays: the microtubule-associated protein tau for degenerated axons and S-100B for astrocytes. The level of tau proteins in the CSF was significantly elevated in children with cerebral malaria compared with either malaria with prostration or malaria with seizures but normal consciousness (p<0.001). Elevated tau was also found to be associated with impaired delivery of oxygen (severe anaemia), severe metabolic acidosis manifesting as respiratory distress (increased respiratory rate and deep acidotic breathing) and at higher parasite densities. Elevated S-100B in children was associated with an increased risk of repeated seizures. This study provides evidence that axonal injury is associated with malaria coma and identifies the potential role of severe anaemia, acidosis and hyperparasitaemia to causing brain parenchymal damage in children with malaria.

Cytotoxic T lymphocytes in autoimmune and degenerative CNS diseases.

Cytotoxic T lymphocytes (CTLs) with a CD8(+) phenotype have the potential to recognize and attack major histocompatibility complex (MHC) class I-expressing brain cells. Most brain cells, including neurons, can be stimulated to present peptides to CD8(+) CTLs by MHC class I molecules, and are susceptible to CTL-mediated cytotoxicity in culture. In disease-affected brain parenchyma, CD8(+) CTLs outnumber other T-cell subtypes. They show clonal expansion in several inflammatory and degenerative CNS diseases, such as multiple sclerosis (MS), virus-induced inflammatory brain diseases and paraneoplastic neurological disorders. In MS, damage of axons is closely linked to the CD8(+) CTLs, and protection against CTL-mediated damage should be considered as a new therapeutic approach in MS and other neuroinflammatory diseases.

Human cerebral malaria and the blood-brain barrier.

Malaria represents a continuing and major global health challenge and our understanding of how the Plasmodium parasite causes severe disease and death remains poor. One serious complication of the infection is cerebral malaria, a clinically complex syndrome of coma and potentially reversible encephalopathy, associated with a high mortality rate and increasingly recognised long-term sequelae in survivors. Research into the pathophysiology of cerebral malaria, using a combination of clinical and pathological studies, animal models and in vitro cell culture work, has focussed attention on the blood-brain barrier (BBB). This represents the key interface between the brain parenchyma and the parasite, which develops within an infected red cell but remains inside the vascular space. Studies of BBB function in cerebral malaria have provided some evidence for parasite-induced changes secondary to sequestration of parasitised red blood cells and host leukocytes within the cerebral microvasculature, such as redistribution of endothelial cell intercellular junction proteins and intracellular signaling. However, the evidence for a generalised increase in BBB permeability, leading to cerebral oedema, is conflicting. As well as direct cell adhesion-dependent effects, local adhesion-independent effects may activate and damage cerebral endothelial cells and perivascular cells, such as decreased blood flow, hypoxia or the effects of parasite toxins such as pigment. Finally, a number of systemic mechanisms could influence the BBB during malaria, such as the metabolic and inflammatory complications of severe disease acting 'at a distance'. This review will summarise evidence for these mechanisms from human studies of cerebral malaria and discuss the possible role for BBB dysfunction in this complex and challenging disease.

Immunopathogenesis of cerebral malaria.

Malaria is 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, yet its pathogenesis remains incompletely understood. In this review, we discuss some of the principal pathogenic events that have been described in murine models of the disease and relate them to the human condition. One of the earliest events in CM pathogenesis appears to be a mild increase in the permeability to protein of the blood-brain barrier. Recent studies have shown a role for CD8+T cells in mediating damage to the microvascular endothelium and this damage can result in the leakage of cytokines, malaria antigens and other potentially harmful molecules across the blood-brain barrier into the cerebral parenchyma. We suggest that this, in turn, leads to the activation of microglia and the activation and apoptosis of astrocytes. The role of hypoxia in the pathogenesis of cerebral malaria is also discussed, with particular reference to the local reduction of oxygen consumption in the brain as a consequence of vascular obstruction, to cytokine-driven changes in glucose metabolism, and to cytopathic hypoxia. Interferon-gamma, a cytokine known to be produced in malaria infection, induces increased expression, by microvascular endothelial cells, of the haem enzyme indoleamine 2,3-dioxygenase, the first enzyme in the kynurenine pathway of tryptophan metabolism. Enhanced indoleamine 2,3-dioxygenase expression leads to increased production of a range of biologically active metabolites that may be part of a tissue protective response. Damage to astrocytes may result in reduced production of the neuroprotectant molecule kynurenic acid, leading to a decrease in its ratio relative to the neuroexcitotoxic molecule quinolinic acid, which might contribute to some of the neurological symptoms of cerebral malaria. Lastly, we discuss the role of other haem enzymes, cyclooxygenase-2, inducible nitric oxide synthase and haem oxygenase-1, as potentially being components of mechanisms that protect host tissue against the effects of cytokine- and leukocyte-mediated stress induced by malaria infection.

Cerebrospinal fluid levels of markers of brain parenchymal damage in Vietnamese adults with severe malaria.

A retrospective study of cerebrospinal fluid (CSF) markers of brain parenchymal damage was conducted in Vietnamese adults with severe malaria. Three markers were analysed by immunoassays: the microtubule-associated protein tau, for degenerated axons; neuron-specific enolase (NSE), for neurons; and S100B for astrocytes. The mean concentration of tau proteins in the CSF was significantly raised in patients with severe malaria compared with controls (P=0.0003) as reported for other central nervous system diseases. By contrast, the mean concentration of NSE and S100B remained within the normal range. Tau levels were associated with duration of coma (P=0.004) and S100B was associated with convulsions (P=0.006). Concentrations of axonal and astrocyte degeneration markers also were associated with vital organ dysfunction. No association was found between the level of markers of brain parenchymal damage on admission and a fatal outcome. On admission to hospital, patients with severe malaria had biochemical evidence of brain parenchymal damage predominantly affecting axons.

The murine cerebral malaria phenomenon.

P.berghei ANKA infection in CBA or CB57BL/6 mice is used widely as a murine 'model' of human cerebral malaria (HCM), despite markedly different histopathological features. The pathology of the murine model is characterised by marked inflammation with little or no intracerebral sequestration of parasitised erythrocytes, whereas HCM is associated with intense intracerebral sequestration, often with little inflammatory response. There are now more than ten times as many studies each year of the murine model than on HCM. Of 48 adjunctive interventions evaluated in the murine model, 44 (92%) were successful, compared with only 1 (6%) of 17 evaluated in HCM during the same period. The value of the mouse model in identifying pathological processes or therapeutic interventions in human cerebral malaria is questionable.

Metabolites of the kynurenine pathway of tryptophan metabolism in the cerebrospinal fluid of Malawian children with malaria.

A retrospective study of 100 Malawian children (87 with malaria and 13 with a diagnosis other than malaria) was conducted to determine the relationship between levels of metabolites of the kynurenine pathway in cerebrospinal fluid (CSF) and disease outcome. Three metabolites were measured: quinolinic acid (QA), an excitotoxin; kynurenic acid (KA), a neuroprotective receptor antagonist; and picolinic acid (PA), a proinflammatory mediator. Elevated levels of QA and PA in CSF were associated with a fatal outcome in Malawian children with cerebral malaria (CM). QA was associated with a history of convulsions. An increase in the QArcolon;KA ratio, which favors neurotoxicity, was observed only in the 3 patients with tuberculosis meningitis. Compared with Vietnamese adults with malaria, Malawian children with malaria had higher concentrations of KA. Elevated levels of KA in children with CM may serve to contain injury in the developing brain, which is more susceptible to excitotoxic damage than is the adult brain.

Axonal injury in cerebral malaria.

Impairment of consciousness and other signs of cerebral dysfunction are common complications of severe Plasmodium falciparum malaria. Although the majority of patients make a complete recovery a significant minority, particularly children, have sequelae. The pathological process by which P. falciparum malaria induces severe but usually reversible neurological complications has not been elucidated. Impairment of transport within nerve fibers could induce neurological dysfunction and may have the potential either to resolve or to progress to irreversible damage. Beta-amyloid precursor protein (beta-APP) immunocytochemistry, quantified using digital image analysis, was used to detect defects in axonal transport in brain sections from 54 Vietnamese cases with P. falciparum malaria. The frequency and extent of beta-APP staining were more severe in patients with cerebral malaria than in those with no clinical cerebral involvement. Beta-APP staining was often associated with hemorrhages and areas of demyelination, suggesting that multiple processes may be involved in neuronal injury. The age of focal axonal damage, as determined by the extent of the associated microglial response, varied considerably within tissue sections from individual patients. These findings suggest that axons are vulnerable to a broad range of cerebral insults that occur during P. falciparum malaria infection. Disruption in axonal transport may represent a final common pathway leading to neurological dysfunction in cerebral malaria.

The clinical significance of cerebrospinal fluid levels of kynurenine pathway metabolites and lactate in severe malaria.

A retrospective study of 261 Vietnamese adults with severe malaria was conducted to determine the relationship between cerebrospinal fluid (CSF) levels of metabolites of the kynurenine pathway, the incidence of neurologic complications, and the disease outcome. Three metabolites were measured: the excitotoxin quinolinic acid (QA); the protective receptor antagonist kynurenic acid (KA); and the proinflammatory mediator picolinic acid (PA). These measurements were related prospectively to CSF lactate levels. QA and PA levels were elevated, compared with those of controls. There was no difference in the levels of KA between these groups. Although >40% of malaria patients had QA CSF concentrations in the micromolar range, there was no association with convulsions or depth of coma. Levels of QA and PA were associated significantly with death, but a multivariate analysis suggested that these elevations were a consequence of impaired renal function. CSF lactate remained an independent and significant predictor of poor outcome.

Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells.

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.