Magnetic resonance spectroscopy reveals an impaired brain metabolic profile in mice resistant to cerebral malaria infected with Plasmodium berghei ANKA

Malaria is a major cause of morbidity and mortality with an annual death toll exceeding one million. Severe malaria is a complex multisystem disorder, including one or more of the following complications: cerebral malaria, anemia, acidosis, jaundice, respiratory distress, renal insufficiency, coagulation anomalies, and hyperparasitemia. Using a combined in vivo/in vitro metabolic-based approach, we investigated the putative pathogenic effects of Plasmodium berghei ANKA on brain, in a mouse strain developing malaria but resistant to cerebral malaria. The purpose was to determine whether the infection could cause a brain dysfunction distinct from the classic cerebral syndrome. Mice resistant to cerebral malaria were infected with P. berghei ANKA and explored during both the symptomless and the severe stage of the disease by using in vivo brain magnetic resonance imaging and spectroscopy. The infected mice did not present the lesional and metabolic hallmarks of cerebral malaria. However, brain dysfunction caused by anemia, parasite burden, and hepatic damage was evidenced. We report an increase in cerebral blood flow, a process allowing temporary maintenance of oxygen supply to brain despite anemia. Besides, we document metabolic anomalies affecting choline-derived compounds, myo-inositol, glutamine, glycine, and alanine. The choline decrease appears related to parasite proliferation. Glutamine, myo-inositol, glycine, and alanine variations together indicate a hepatic encephalopathy, a finding in agreement with the liver damage detected in mice, which is also a feature of the human disease. These results reveal the vulnerability of brain to malaria infection at the severe stage of the disease even in the absence of cerebral malaria.     

Plasmodium falciparum: polymorphism in the MSP-1 gene in Indian isolates and predominance of certain alleles in cerebral malaria

Polymorphism in the block-2 region of merozoite surface protein-1 gene in 69 North Indian Plasmodium falciparum isolates was studied by PCR and RFLP using Dra-1 endonuclease. On the basis of molecular weight of the PCR products, considerable size polymorphism in target gene was seen and 69 isolates were classified into five allelic types. On RFLP, the isolates in three allelic types were further divided into two sub-allelic types each and thus eight genetic types could be identified. Interestingly, all five allelic types were identified in 47 isolates from uncomplicated (non-cerebral) malaria patients while only two allelic types (Type 2 and 3) were seen amongst 22 isolates from cerebral malaria patients. Furthermore, on RFLP, one subtype (2A) was predominantly seen in cerebral malaria patients and one subtype (3A) was exclusively found in cerebral malaria patients. These observations suggest that a few, comparatively more virulent isolates prevalent in an area may cause severe disease (cerebral malaria) which can be identified by molecular techniques like PCR-RFLP.     

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.     

Brain metabolic markers reflect susceptibility status in cytokine gene knockout mice with murine cerebral malaria

Treatment of cerebral malaria, a complication of the world's most significant parasitic disease, remains problematic due to lack of understanding of its pathogenesis. Metabolic changes, along with cytokine expression alterations and blood cell sequestration in the brain, have previously been reported during severe disease in human infection and mouse models leading to the "cytopathic hypoxia" and "sequestration" theories of pathogenesis. Here, to determine the robustness of the metabolic changes and their relationship to disease development, we investigated changes in cerebral metabolic markers in a mouse model of cerebral malaria (CM) in wildtype (C57BL/6) and cytokine knockout (TNF(-/-), IFNgamma(-/-) and LTalpha(-/-)) mice using multinuclear magnetic resonance spectroscopy. Mice susceptible to CM (wildtype, TNF(-/-)) showed decreased cerebral glucose use, decreased Krebs cycle metabolism and decreased high-energy phosphates. Conversely, mice resistant to CM (IFNgamma(-/-), LTalpha(-/-)) showed little sign of these effects, despite identical levels of parasitemia. Previously reported changes in lactate were shown to be strain dependent. Elevated glutamine and decreased phosphorylation potential emerged as robust metabolic markers of susceptibility, further implicating the trytophan/NAD(+) pathway in disease development. Thus these metabolic changes are firmly linked both to the immune system response to malaria and to the occurrence of pathogenic changes in experimental CM.     

Breaking down the blood-brain barrier: signaling a path to cerebral malaria?

Cerebral malaria is a major killer in the developing world, but we still know very little about the causes of this disease. How does Plasmodium falciparum cause such a devastating neurological disease while it is in the brain vasculature? Why do some patients die, whereas others survive? What processes contribute to disease in the brain, and can we reverse them? Here, the latest evidence from post-mortem, in vitro and animal studies is reviewed to highlight the role of blood-brain barrier breakdown in cerebral malaria. Blood-brain barrier integrity is disturbed during severe malaria, causing leakage of cerebral vessels. Understanding how this happens and how it contributes to the pathogenesis of coma may provide new opportunities for the treatment of cerebral malaria.     

Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity

In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area, whereas recognition of VSA expressed by parasites from older children was weaker and less frequent. Independent of this, parasites isolated from children with severe malaria (cerebral malaria and severe anemia) were better recognized by VSA-specific plasma Abs than parasites obtained from children with nonsevere disease. This was not due to a higher infection multiplicity in younger patients or in patients with severe disease. Our data suggest that acquisition of VSA-specific Ab responses gradually restricts the VSA repertoire that is compatible with parasite survival in the semi-immune host. This appears to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random, and that the VSA expression pattern is modulated by immunity. This opens the possibility of developing morbidity-reducing vaccines targeting a limited subset of common and particularly virulent VSA.     

The plant-based immunomodulator curcumin as a potential candidate for the development of an adjunctive therapy for cerebral malaria

The clinical manifestations of cerebral malaria (CM) are well correlated with underlying major pathophysiological events occurring during an acute malaria infection, the most important of which, is the adherence of parasitized erythrocytes to endothelial cells ultimately leading to sequestration and obstruction of brain capillaries. The consequent reduction in blood flow, leads to cerebral hypoxia, localized inflammation and release of neurotoxic molecules and inflammatory cytokines by the endothelium. The pharmacological regulation of these immunopathological processes by immunomodulatory molecules may potentially benefit the management of this severe complication. Adjunctive therapy of CM patients with an appropriate immunomodulatory compound possessing even moderate anti-malarial activity with the capacity to down regulate excess production of proinflammatory cytokines and expression of adhesion molecules, could potentially reverse cytoadherence, improve survival and prevent neurological sequelae. Current major drug discovery programmes are mainly focused on novel parasite targets and mechanisms of action. However, the discovery of compounds targeting the host remains a largely unexplored but attractive area of drug discovery research for the treatment of CM. This review discusses the properties of the plant immune-modifier curcumin and its potential as an adjunctive therapy for the management of this complication.     

Clinical Investigation of Fasudil for the Prevention of Cerebral Vasospasm in Extracranial Carotid Artery Stenting

To evaluate fasudil hydrochloride for the prevention of cerebral vasospasm (CVS) in extra-cranial carotid angioplasty and stenting (CAS). We retrospectively analyzed 178 patients with unilateral CAS who were given intravenous fasudil hydrochloride during the perioperative period. CVS, hypotension, stroke, and mortality incidence rates were recorded. Of the cohort studied, 80.9 % patients exhibited no local CVS, asymptomatic vasospasm was observed in 17.4 % patients and symptomatic vasospasm in 1.7 % patients via DSA imaging. All CVS was relieved and symptoms disappeared after intra-arterial infusion of papaverine hydrochloride. Intracerebral hemorrhage occurred in two cases during the perioperative period, one of which resulted in death. CVS is a severe complication of CAS. Fasudil hydrochloride can rapidly relieve cerebral vasospasm, has no selective effect on cerebral vasculature, and little influence on blood pressure. It is suitable for the prevention of CVS during interventional treatment of ischemic cerebrovascular disease.     

Malaria transmission and morbidity

Stable malaria endemicity is maintained over a wide range of transmission intensities in sub-Saharan Africa. This paper considers variations in the clinical manifestations and their consequences with differences in transmission intensity. Epidemiological approaches to malarial disease have concentrated on two clinical syndromes, severe malarial anaemia and cerebral malaria. Within an area the mean age of children with severe malarial anaemia is always lower than that of those with cerebral malaria. In areas of higher malaria transmission children, on average, encounter malaria at a younger age and the mean age of clinical cases is lower. Malarial anaemia tends therefore to be relatively more important under high transmission settings and cerebral malaria tends to gain in importance under lower transmission settings. In a number of studies the total load of malaria morbidity, whether measured as none severe malaria in the community or as severe malaria admitted to hospital, is low under stable low transmission conditions but is at its highest under moderate intensities of transmission. Thereafter it reaches a plateau, or even falls, at the highest transmission intensities. It is not known whether the same is true for mortality in communities living under different transmission settings. Possible implications for changes in patterns of morbidity and mortality following interventions which lower malaria transmission are discussed. It is concluded that such interventions should play an important role in integrated malaria control programmes but that these should involve concomitant introduction of other interventions, in order to minimise the possible risks of a reduced effect as the immune response of the population re-equilibrates in the face of reduced challenge.     

Acquired immunity and postnatal clinical protection in childhood cerebral malaria

By analysing data on the age distribution of cerebral malaria among sites of different transmission intensities, we conclude that the most plausible explanation for the epidemiological patterns seen is that (i) cerebral malaria is caused by a distinct set of Plasmodium falciparum antigenic types; (ii) these antigenic types or 'CM strains' are very common and induce strong strain-specific immunity; and (iii) the postnatal period of protection against cerebral malaria is much longer than the period of protection against other forms of severe disease. The alternative hypothesis that cerebral malaria may be caused by any 'strain' of P. falciparum is compatible with the data only if a single exposure is sufficient to protect against further episodes. This is not consistent with observations on the history of exposure of patients with cerebral malaria. Finally, it is clear that although the delayed peak in incidence of cerebral malaria (with age) can be generated by assuming that subsequent exposures carry a higher risk of disease, such an explanation is not compatible with the observation that severe disease rates are low among infants and young children in areas of high transmissibility.     

Neuroprotective effects of NEP1-40 and fasudil on Nogo-A expression in neonatal rats with hypoxic-ischemic brain damage

The hypoxic-ischemic encephalopathy caused by peripartum asphyxia is a serious disease in newborn infants, and effective therapies need to be developed to reduce injury-related disorders. We evaluated the effects of NEP1-40 and fasudil on Nogo-A expression in neonatal hypoxic-ischemic brain damage (HIBD) rats. Seven-day-old Wistar rats were randomly divided into control, HIBD, NEP1-40, and fasudil groups. NEP1-40 and fasudil groups were injected intraperitoneally with these compounds. Rat brains at 6, 24, 72 h, and 7 days after HIBD were collected to determine histopathological damage and the expression levels of Nogo-A. Histopathological damage was reduced in NEP1-40 and fasudil groups compared with the untreated HIBD group. The expression of Nogo-A in the HIBD group was significantly higher than that in control, NEP1-40 and fasudil groups at the same times. Compared with the fasudil group, the expression levels of Nogo-A were significantly reduced in the NEP1-40 group. We conclude that NPE1-40 and fasudil have potential for neuroprotective effects in the neonatal rat HIBD model, mediated by inhibiting Nogo-A/ Rho pathways.     

Applications for ROCK kinase inhibition

ROCK kinases, which play central roles in the organization of the actin cytoskeleton, are tantalizing targets for the treatment of human diseases. Deletion of ROCK I in mice revealed a role in the pathophysiological responses to high blood pressure, and validated ROCK inhibition for the treatment of specific types of cardiovascular disease. To date, the only ROCK inhibitor employed clinically in humans is fasudil, which has been used safely in Japan since 1995 for the treatment of cerebral vasospasm. Clinical trials, mostly focusing on the cardiovascular system, have uncovered beneficial effects of fasudil for additional indications. Intriguing recent findings also suggest significant potential for ROCK inhibitors in the production and implantation of stem cells for disease therapies.     

The effect of helminth co-infection on malaria in mice: a meta-analysis

The question of how helminths may alter the course of concurrent malaria infection has attracted much interest in recent years. In particular, it has been suggested that by creating an anti-inflammatory immune environment, helminth co-infection may dampen both protective and immunopathological responses to malaria parasites, thus altering malaria infection dynamics and disease severity. Both synergistic and antagonistic interactions are reported in the literature, and the causes of variation among studies are not well understood. Here, meta-analysis of 42 mouse co-infection experiments was used to address how helminths influence malaria parasite replication and host mortality, and explore the factors explaining variation in findings. Most notably, this analysis revealed contrasting effects of helminth co-infection in lethal and resolving malaria models. Whilst co-infection exacerbated mortality and increased peak parasitaemia in ordinarily resolving malaria infections (Plasmodium chabaudi and Plasmodium yoelii), effects among lethal malaria infections (Plasmodium berghei) tended to be in the opposite direction with no change in parasitaemia. In the subset of experiments on cerebral malaria models (P. berghei ANKA strain in a susceptible host), helminth co-infection significantly delayed death. These findings are consistent with the hypothesis that depending on the existing balance of pro- and anti-inflammatory responses mounted against malaria parasites in a given host, immune responses elicited by helminth co-infection may either promote or inhibit malarial disease. However, despite such broad patterns, a prominent feature of this dataset was great heterogeneity in effects across studies. A key future challenge therefore lies in explaining the biological causes of this variation, including a more thorough exploration of non-immunological mechanisms of helminth-malaria interaction.     

Imported Malaria in Korea: a 13-Year Experience in a Single Center

The incidence of imported malaria has been increasing in Korea. We reviewed data retrospectively to evaluate the epidemiology, clinical features, and outcomes of imported malaria from 1995 to 2007 in a university hospital. All patients diagnosed with imported malaria were included. Imported malaria was defined as a positive smear for malaria that was acquired in a foreign country. A total of 49 patients (mean age, 35.7 year; M : F = 38 : 11) were enrolled. The predominant malarial species was Plasmodium falciparum (73.5%), and the most frequent area of acquisition was Africa (55.1%), followed by Southeast Asia (22.4%) and South Asia (18.4%). Fourteen-patients (30.6%) suffered from severe malaria caused by P. falciparum and 1 patient (2.0%) died of multiorgan failure. Most of the patients were treated with mefloquine (79.2%) or quinine (10.2%); other antimalarial agents had to be given in 13.2% treated with mefloquine and 44.4% with quinine due to adverse drug events (ADEs). P. falciparum was the most common cause of imported malaria, with the majority of cases acquired from Africa, and a significant number of patients had severe malaria. Alternative antimalarial agents with lower rates of ADEs might be considered for effective treatment instead of mefloquine and quinine.     

Curcumin Improves Outcomes and Attenuates Focal Cerebral Ischemic Injury via Antiapoptotic Mechanisms in Rats

Curcumin, a member of the curcuminoid family of compounds, is a yellow colored phenolic pigment obtained from the powdered rhizome of C. longa Linn. Recent studies have demonstrated that curcumin has protective effects against cerebral ischemia/reperfusion injury. However, little is known about its mechanism. In the present study, we tested the effects of curcumin in focal cerebral ischemia in rats and the possible mechanisms. Adult male Sprague–Dawley rats were treated with curcumin (100, 300 and 500 mg/kg) administered intraperitoneally after 60 min of occlusion (beginning of reperfusion). Neurological score and infarct volume were assessed at 24 and 72 h. Oxidative stress was evaluated by malondialdehyde assay and the apoptotic mechanisms were studied by Western blotting. Curcumin treatment significantly reduced infarct volume and improved neurological scores at different time points compared with the vehicle-treated group. Curcumin treatment decreased malondialdehyde levels, cytochrome c, and cleaved caspase 3 expression and increased mitochondrial Bcl-2 expression. Inhibition of oxidative stress with curcumin treatment improves outcomes after focal cerebral ischemia. This neuroprotective effect is likely exerted by antiapoptotic mechanisms.     

Pharmacological profile of hydroxy fasudil as a selective rho kinase inhibitor on ischemic brain damage

The neuroprotective property and the effects on hemodynamics of hydroxy fasudil, an active metabolite of an antispastic drug, fasudil, were examined. In rats, hydroxy fasudil was found following intravenous infusion or intraperitoneal administration of fasudil, and the maximum plasma concentration of hydroxy fasudil was approximately 25 or 40% of the parent drug, respectively. The i.v. administration of hydroxy fasudil produced significant increases in regional cerebral blood flow in dogs. Hydroxy fasudil relaxed the KCl, PGF2alpha or U-46619-induced contraction in canine basilar or middle cerebral arterial strips, concentration-dependently. The neuroprotective property of hydroxy fasudil was examined on delayed neuronal death in gerbils. Hydroxy fasudil (3 mg/kg) significantly protected against the ischemia-induced neuronal loss. To further clarify the effect on neurological impairments, hydroxy fasudil was tested in a rat model of microembolization stroke. Intravenous administration of hydroxy fasudil improved neurological functions, significantly reduced the size of the infarct area and prevented the accumulation of neutrophils. The present findings suggest that hydroxy fasudil has an efficacy to improve the hemodynamic function and to inhibit neutrophil-mediated damage, and contributes to the potency and long duration of the cytoprotective properties of fasudil on ischemic brain damage, and also suggest a critical role for rho kinase in the pathogenesis of cerebral ischemic injury, and the potential utility of rho kinase inhibitor as a therapeutic agent in stroke.     

Severe malaria: lessons learned from the management of critical illness in children

Two hypotheses have recently been raised to explain the metabolic acidosis (increased blood acidity) of severe malaria, and both are relevant to treatment. The first suggests that a decreased blood volume (hypovolaemia) has an important role in severe malaria; following this, treatment should be based on the current standard paediatric management of acidosis in children with features of cardiovascular compromise. The second hypothesis contends that acidosis in malaria has a metabolic cause and proposes treatment with dichloroacetate. Both hypotheses are plausible and are not mutually exclusive. In truth, the risks and benefits of either treatment are uncertain, and will remain so until large multicentre, randomised controlled trials provide appropriate supportive evidence. As both views involve complex physiological rationales, beyond the usual scope of this journal, I attempt here to present the largely academic aspects of these hypotheses within the practical and contextual aspects of childhood severe malaria.     

TNF family members and malaria: Old observations, new insights and future directions

Tumor necrosis factor (TNF) has long been recognized to promote malaria parasite killing, but also to contribute to the development of severe malaria disease. The precise molecular mechanisms that influence these different outcomes in malaria patients are not well understood, but the virulence and drug-resistance phenotype of malaria parasites and the genetic background and age of patients are likely to be important determinants. In the past few years, important roles for other TNF family members in host immune responses to malaria parasites and the induction of disease pathology have been discovered. In this review, we will summarize these more recent findings and highlight major gaps in our current knowledge. We will also discuss future research strategies that may allow us to better understand the sometimes subtle and intricate effects of TNF family molecules during malaria infection.     

Malaria-related health-seeking behaviour and challenges for care providers in rural Ethiopia: implications for control

A range of activities are currently underway to improve access to malaria prevention and control interventions. As disease control strategies change over time, it is crucial to understand the health-seeking behaviour and the local socio-cultural context in which the changes in interventions operate. This paper reflects on how people in an area of seasonal malaria perceive the causes and transmission of the disease, and what prevention and treatment measures they practise to cope with the disease. It also highlights some of the challenges of malaria treatment for health care providers. The study was undertaken in 2003 in Adami Tulu District in south-central Ethiopia, where malaria is a major health problem. Pre-tested structured questionnaires and focus group discussions were conducted among men and women. Malaria, locally known as busa, was perceived as the most important cause of ill health in the area. Respondent's perception and knowledge about the cause and transmission of the disease were relatively high. The newly introduced insecticide-treated nets were not popular in the area, and only 6.4% of households possessed at least one. The results showed that patients use multiple sources of health care for malaria treatment. Public health facilities, private clinics and community health workers were the main providers of malaria treatment. Despite higher treatment costs, people preferred to use private health care providers for malaria treatment due to the higher perceived quality of care they offer. In conclusion, effort in the prevention and control of malaria should be intensified through addressing not only public facilities, but also the private sector and community-based control interventions. Appropriate and relevant information on malaria should be disseminated to the local community. The authors propose the provision of effective antimalarial drugs and malaria prevention tools such as subsidized or free insecticide-treated nets.