Cerebral Malaria: A Vasculopathy

This Commentary highlights recent advances in research on cerebral malaria.Cerebral malaria (CM), an important disease entity in the developing world, remains the deadliest complication of infection with P. falciparum. According to a recent World Health Organization report, half of the world''s population remains at risk of acquiring malaria. The majority of the burden of disease occurs in Sub-Saharan Africa, where children under the age of five account for more than 80% of malaria-related deaths. CM is associated with an encephalitic syndrome that includes ataxia, seizures, hemiplegia, and eventually coma and death. These symptoms of severe malaria can progress very precipitously within hours from mild to severe. Indeed, even with successful antiparasitic treatment, residual neurological damage is a common finding in CM. It is estimated that more than 10% to 20% of the children who survive an episode of CM develop long-term cognitive deficits, which can include memory impairment, learning and language impairments, visuospatial and motor deficits, and psychiatric disorders.The etiology of CM is not entirely understood because clinical studies in humans are not always feasible, and autopsy studies have only given us a limited view of this disease syndrome. In recent years, the increasing availability of computerized axial tomography (CT) and magnetic resonance imaging (MR) scanning has increased our understanding of the pathophysiology of this entity. Multiple mechanisms have been proposed to govern CM pathogenesis, including an intense upregulation of the inflammatory response, hypoxia, hypoglycemia, monocytic and/or red blood cell sequestration, and microvascular dysfunction leading to ischemia. The etiology of CM is likely multifactorial, and these hypotheses are not mutually exclusive.The mouse model of CM using C57BL/6 mice infected with the ANKA strain of P. berghei as described by Cabrales et al¹ in the current issue of this Journal recapitulates many of the features of human CM. Although there has been some discussion as to whether this particular mouse model is a reliable model of human CM, it is now generally acknowledged to be a relevant and practical small animal model for CM. The pathological features of both human CM and the murine model described here and by others include microhemorrhages and vascular occlusion. However, the nature of the vascular occlusion in murine CM differs from that observed in human CM in that the former displays no red blood cell adherence and/or occlusion. Importantly, cognitive dysfunction has been observed in this animal model.²Recently, a number of studies implicate a disruption in the integrity of the cerebral vasculature as an important contributing factor in the pathogenesis of CM. Both human and experimental CM studies are associated with a reduction in cerebral blood flow (CBF), which may be an important factor in the progression to CM. Single photon emission computed tomography (SPECT) in human CM demonstrated marked cerebral hypoperfusion associated with a significant decrease in oxygen saturation and neurological deficits corresponding to the areas of hypoperfusion.^3,4 These abnormalities include decreased or absent perfusion in the capillaries and in larger retinal vessels, intravascular filling defects and leakage of dye material, which is indicative of a breakdown of the blood–retinal barrier, and ischemia.⁵ The ischemic changes often correlate with neurological sequelae including seizures, obtundation, and coma.In the current issue of the Journal, Cabrales et al¹ present substantial evidence for a role for vasoconstriction in the setting of CM and highlight the importance of vascular dysfunction in the pathogenesis of CM. Through the use of intravital microscopy, these authors obtained direct visualization of the pial microvasculature of the brain and correlated vascular dysfunction with progression of CM. Importantly, this disease progression was reversed when the vasculopathy was corrected by the calcium-channel blocker nimodipine.Previously, it was demonstrated that in the murine model of CM, a reduction in CBF at advanced stages of the disease as measured by MRI/MRA directly correlated with significant decreases in the levels of certain metabolic markers in areas of the brain that were indicative of neuronal damage.⁵ Specifically, a decrease in CBF was reported to be associated with a reduction in the ratio of N-acetyl aspartate (NAA) to creatine.⁵ NAA has been widely used as an inverse marker of neuronal loss and injury in a variety of pathologies. It is synthesized almost exclusively in neuronal mitochondria, and a decrease in NAA levels usually reflects a mixture of both neuronal loss and recent or ongoing neuronal injury/dysfunction. A reduction in cerebral perfusion has also been associated with damage in the neuron/axon compartment with CM.⁵ Conversely, MR spectroscopy studies of mice resistant to murine CM demonstrated no change in CBF or metabolic profile and no central nervous system lesions. These data indicate that alterations in the vasculature are an important component of CM.In the present report, Cabrales et al¹ demonstrated a clear correlation with neurological deficits such as ataxia, limb paralysis, poor righting reflex, and seizures and the changes in the pial vessels. These deficits appear to be lesion-dependent, as mice with more severe neurological symptoms had a greater degree of vascular constriction and even sustained complete vascular collapse, whereas those with no signs of CM had a minimal decrease in CBF. Importantly, treatment with nimodipine together with the antimalarial agent artemether not only resulted in improved survival but also in a more rapid return to normal neurological function. The authors suggest that the reason for this observation is the partial restoration of CBF in affected mice.The vasculopathy associated with CM is likely a result of endothelial cell damage, ischemia, activation of vascular cell adhesion molecules, and an associated breakdown in the blood–brain barrier.^6,7 Recently, we have focused on the role of vasoactive compounds in the setting of CM, particularly the 21-aa vasopeptide endothelin (ET-1).⁸ Elevated plasma levels of ET-1 and big ET-1 have been reported in patients with P. falciparum. This occurs during acute infection and persists days after treatment of malaria. The increase in ET-1 correlates with elevated levels of TNF-α and likely reflects damage to the endothelium. A similar observation has been reported in an experimental CM model. In this same mouse model, there was an increase in the expression of ET-1 and endothelin converting enzyme, the enzyme that is responsible for the synthesis of ET-1 from Big ET-1, as well as increased expression of the endothelin receptors (ET_A and ET_B). This increase in the components of the endothelin pathway was associated with a reduction in CBF and neuronal dysfunction and inflammation.⁸ The increase in ET-1 in the brain of mice with CM is consistent with the findings of increased plasma levels of big ET-1 in patients with acute complicated disease. Interestingly, the intraventricular injection of ET-1 has been reported to result in behavioral changes, including barrel rolling, body tilting, nystagmus, clonus, and tail extension. Most of these effects occur at doses that do not cause any changes in CBF.Low levels of nitric oxide (NO) derived from endothelial nitric oxide synthase (NOS) and neuronal NOS have also been implicated in the vasculopathy of CM, further supporting the observation that vasoconstriction is an integral part in the development of CM. Cabrales et al¹ suggest that microhemorrhages may be important in this disease process because of the alterations in NO levels, which are known to be associated with cerebral hemorrhages.Using calcium-channel blockers to ameliorate vascular spasm is not entirely novel in experimental cardiovascular disease. For example, Factor et al⁹ demonstrated that the administration of verapamil to Syrian cardiomyopathic hamsters ameliorated the vasospasm of the coronary microvasculature, thus resulting in improvement of the cardiomyopathic phenotype. When verapamil was administered early, but not late, in the course of experimental Trypanosoma cruzi infection, it prevented the appearance of cardiomyopathy.¹⁰ Furthermore, Tanowitz et al¹¹ used a cremaster muscle preparation to demonstrate that the T. cruzi--associated vasospasm was prevented by the administration of verapamil, similar to the findings in CM in the current report with nimodipine.¹ Nimodipine, the calcium-channel blocker used in the present study, ameliorates the vasospasm accompanying subarachnoid hemorrhage.¹Drugs of seemingly disparate classes show some efficacy in experimental CM. For example, Serghides et al¹² recently reported that the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist rosiglitazone improved the outcome in CM in the mouse model. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HMG-CoA reductase inhibitor or statin) atorvastatin has been shown to be highly efficacious when used in combination with artesunate, resulting in a significant decrease in mortality.¹³ Erythropoietin has also been reported as effective in ameliorating CM.¹⁴ These agents are used clinically because of their effects on cholesterol metabolism, insulin resistance, and erythropoiesis, respectively. Pertinent to the current study, however, they share with calcium-channel blockers the ability to modulate the repair of microvascular damage.Damage to the microvasculature, as elegantly demonstrated in the current study, is generally repaired either by replication of local existing endothelial cells, or by bone marrow–derived circulating endothelial progenitor cells (cEPCs), which are stimulated to migrate and incorporate into damaged sites in the microvasculature.¹⁵ Their mobilization, release, and ultimate incorporation into sites of microvascular damage are mediated by the activation of a series of molecules, including stromal cell derived growth factor 1 (SDF-1). The subsequent incorporation of cEPCs into sites of microvascular damage is essential to the maintenance of microvascular integrity. They play a critical role in diseases associated with chronic or extensive damage. Indeed, insufficient or dysfunctional cEPCs and levels of associated factors that effect cEPC mobilization and function are predictive of poor outcomes in diseases associated with microvascular damage such as in cardiovascular disease and stroke^16,17.PPAR-γ agonists, HMG-CoA reductase inhibitors (statins), erythropoietin and calcium channel blockers have all been reported to modulate the cEPC response. PPAR-γ agonists, which promote the differentiation and mobilization of cEPCs, have been shown to promote revascularization postangioplasty and are being evaluated in the treatment of stroke and ischemia/reperfusion injuries.¹⁸ Statins increase cEPC mobilization, numbers, and functional activity, as well as delay cEPC aging and onset of senescence. These effects are independent of their ability to lower cholesterol and are believed to be responsible for the ability of statins to reduce myocardial and cerebral ischemia and cardiovascular risk.¹⁹ Erythropoietin also increases the number of cEPCs in the bone marrow as well as the peripheral circulation. It improves vascularization in murine models of ischemia and has been shown to be safe and effective in the treatment of stroke patients.²⁰ Finally, calcium-channel blockers increase cEPC levels, promote their differentiation and migration as well as their vasodilatory effects. They have therefore been proposed as therapeutic agents to enhance microvascular repair by preserving endothelial cell integrity.²¹A recent study of children with CM in Ghana suggests that the cEPC response to malaria-induced microvascular damage might also play a role in the pathogenesis of CM.²² Children with CM have lower levels of cEPCs as compared with children with uncomplicated malaria, asymptomatic parasitemic children, children with severe malarial anemia, and healthy controls. In addition, levels of SDF-1 are elevated in children with acute disease (uncomplicated malaria and CM), indicative of host attempts to mobilize cEPCs to sites of microvascular damage. These findings place CM within in the context of current paradigms for microvascular repair mechanisms and suggest that equilibrium exists between malaria-induced microvascular damage and host-mediated repair. This balance is maintained by bone marrow–derived cEPCs, which augment local microvascular repair mechanisms in the brain of P. falciparum-infected children.Acute malarial infection is associated with an increase in SDF-1 levels, leading to mobilization and homing of cEPCs to sites of microvascular damage. Thus, hosts able to maintain the balance between damage and repair do not develop CM. If there are low or insufficient numbers of cEPCs to mediate repair, the equilibrium is lost. CM develops, in part, because of breaches in the integrity of the brain microvasculature. Children able to reestablish equilibrium recover, whereas those that are unable to do so likely will die. As described in other diseases associated with microvascular damage, this equilibrium may be lost either because of the exhaustion of bone marrow progenitor cells or because of cEPC dysfunction stemming from reduced migratory capacity, survival, and/or differentiation.²³ Thus, chemotherapeutic agents such as PPAR-γ agonists, statins, erythropoietin, and calcium-channel blockers that encourage microvascular repair could be effective in the prevention and/or treatment of CM. This is consistent with the present study and highlights the role of the microvascular axis as an important target for adjuvant therapies. The demonstration that vasospasm with post-subarachnoid hemorrhage is ameliorated by nimodipine used in this study supports the notion that the host response to the microvascular damage induced by malaria is similar to that of other diseases involving microvasculature dysfunction. This is supported by current paradigms of microvascular repair, which invoke common repair mechanisms and physiological responses regardless of the initial insult to the vasculature.As suggested by the authors, nimodipine, which has been used safely and effectively in humans in the treatment of diseases associated with microvascular damage, may be useful as an adjunctive therapy in the treatment of human CM. The authors are understandably cautious regarding the need for further study of such agents and their effects in murine models of CM attributable to the heterogeneity of case presentations of CM and the multifactorial nature of the disease before clinical trials in humans. The pleomorphic effects of these drugs also require a comprehensive study of their mechanisms of action in malaria using enhanced models such as that described by Cabrales et al.¹For example, the finding that vasodilatation may precede vasoconstriction as observed in some mice with CM might indicate that calcium-channel blockers could be detrimental depending on the time of administration. Indeed in other microvasculature diseases the potential need for “cocktails” of different agents affecting the microvascular axis has been raised with the possibility of both beneficial and detrimental effects. Optimization of murine models of CM permits the direct study of the effects of these agents before use in humans and therefore is an important advance in the field. However, the apparent safety of several modulators of the response to microvascular damage in recent clinical trials in patients with malaria is encouraging.^24,25Finally, it is important to reiterate that in the future, the treatment of CM may involve some form of adjuvant therapy in addition to a potent antimalarial drug. The inclusion of adjuvants, which maintain the integrity of the vasculature, promises to be an important addition to the malaria pharmacopoeia, a point underscored by Carbrales et al.¹     

Toxoplasma gondii Upregulates Interleukin-12 To Prevent Plasmodium berghei-Induced Experimental Cerebral Malaria

A chronic infection with the parasite Toxoplasma gondii has previously been shown to protect mice against subsequent viral, bacterial, or protozoal infections. Here we have shown that a chronic T. gondii infection can prevent Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. Treatment with soluble T. gondii antigens (STAg) reduced parasite sequestration and T cell infiltration in the brains of P. berghei-infected mice. Administration of STAg also preserved blood-brain barrier function, reduced ECM symptoms, and significantly decreased mortality. STAg treatment 24 h post-P. berghei infection led to a rapid increase in serum levels of interleukin 12 (IL-12) and gamma interferon (IFN-γ). By 5 days after P. berghei infection, STAg-treated mice had reduced IFN-γ levels compared to those of mock-treated mice, suggesting that reductions in IFN-γ at the time of ECM onset protected against lethality. Using IL-10- and IL-12βR-deficient mice, we found that STAg-induced protection from ECM is IL-10 independent but IL-12 dependent. Treatment of P. berghei-infected mice with recombinant IL-12 significantly decreased parasitemia and mortality. These data suggest that IL-12, either induced by STAg or injected as a recombinant protein, mediates protection from ECM-associated pathology potentially through early induction of IFN-γ and reduction in parasitemia. These results highlight the importance of early IL-12 induction in protection against ECM.     

Cloned Lines of Plasmodium berghei ANKA Differ in Their Abilities To Induce Experimental Cerebral Malaria

Infection with Plasmodium berghei ANKA is usually lethal. The parasite causes in some mouse strains a neurovascular syndrome, experimental cerebral malaria (ECM), involving immunopathological reactions. The effects on the development of ECM of the mouse genetic background have been clearly demonstrated, but nothing is known about the effects of the clonal diversity of the parasite. We showed that various cloned lines derived from a polyclonal line of P. berghei ANKA caused ECM but that the extent of ECM induction was dependent on the amount of inoculum. Subtle differences in ECM characteristics (survival time and hypothermia) were also observed. We also confirmed, using the 1.49L cloned line, that the mouse genetic background strongly affects ECM.     

急性缺血性脑卒中患者超敏C-反应蛋白和炎性细胞因子水平变化及临床意义

目的:通过检测急性缺血性脑卒中(IMS)患者血清超敏C-反应蛋白(hs-CRP)、肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量的变化,探讨上述炎性介质及炎性细胞因子对IMS的检测价值。方法:选择发病24h内住院的IMS患者68例,并按神经功能缺损评分方法分为轻度缺损组(A组,n=30)、中度缺损组(B组,n=22)和重度缺损组(C组,n=16)。选取30例体检无急性缺血性脑卒中者作对照组,检测血清TNF-α、IL-6和hs-CRP水平。结果:IMS患者血清hs-CRP、TNF-α和IL-6水平均明显高于正常对照组(P<0.05);A组、B组和C组较正常对照组均有不同程度的增高,且随患者神经损害程度的加重而显著升高(P<0.05或P<0.01)。结论:血清hs-CRP和炎性因子TNF-α和IL-6水平可作为IMS患者病情评估的有效指标。     

Analysis by in Situ Hybridization of Cells Expressing mRNA for Tumor-Necrosis Factor in the Developing Thymus of Mice

We have used in situ hybridization to investigate the expression of TNF-α genes by thymic cells during fetal development in mice. In 14-day-old fetal thymuses, very scarce cells produce TNF-α mRNA. A second phase of cytokine gene expression starts on day 16. The density of positive cells progressively increases up to day 20. Thymuses at 15 days of gestation and after birth do not express detectable cytokine mRNA. In an attempt to identify the nature of the TNF-α mRNA-producing cells, acid phosphatase activity, which is characteristic of the macrophage lineage, was studied in the same thymuses. Acid phosphatase-positive cells only appear on day 15. Their frequency increases up to birth. However, no correlation can be established between acid phosphatase—and TNFα mRNA— positive cells. The results indicate that a small subset of thymic cells is responsible for TNF-α mRNA production during ontogeny: These cells are not yet identified. The possible role of TNF-α in thymic ontogeny is discussed.     

青蒿琥酯治疗小儿脑型疟疾48例

目的探讨青蒿琥酯治疗小儿脑型疟疾的疗效。方法脑型疟疾患儿89例，随机分成治疗组（48例）和对照组（41例）；治疗组给予青蒿琥酯，对照组给予奎宁，观察7d后的疗效。结果7d治愈率治疗组为91．67％，对照组为85．37％，差异无统计学意义（P〉0．05）；两组的退热时间分别为（26．1±10．2）h和（39．5±11．6）h，昏迷苏醒时间分别为（36．2±10．1）h和（59．7±12．5）h，差异均有统计学意义（P〈0．01）。对照组的副作用相对较大。结论青蒿琥酯治疗脑型疟疾疗效好而迅速，副作用少，可以作为首选药。     

Cytokines, chemokines and other effector molecules involved in meningococcal disease

This review examines the role of cytokines and chemokines in the pathogenesis of meningococcal disease (MCD) and draws comparisons with studies of other forms of sepsis in adults and in animal models. There are many similarities but also discrepancies between these data. MCD is a well-defined clinical syndrome with identifiable onset and time of presentation. It is a reliable model in which to study cytokine and chemokine responses in bacterial sepsis. Such studies may lead to new adjunctive treatments, which can be tested to ameliorate severe MCD.     

Effects of Pregnancy‐associated Malaria on T Cell Cytokines in Cameroonian Women

Although Th17 cells subsets improve immunity against extra and intracellular pathogens, and in modulating Th1 and other immune responses, its role on pregnancy‐associated malaria (PAM) is unknown. This study aims to investigate the effects of PAM on Th1 (IFN‐γ, TNF‐α), IL‐10 family (IL‐10, IL‐19, IL‐22), Th17 (IL‐17A, IL‐23) cytokines and on CXCL‐10 chemokine profiles in pregnant women. Between 2010 and 2011, venous blood specimens from 107 volunteer pregnant Cameroonian women was used to determine parasitaemia microscopically and haemoglobin levels using HemoCue analyzer. Plasma levels of the biomarkers were determined by ELISA. Parasitaemia was higher in women with low haemoglobin levels, parity and mother's age. IL‐10 and CXCL‐10 plasma levels were higher in the malaria infected and in anaemic women while IFN‐γ and IL‐17A levels were higher in malaria non‐infected and in non‐anaemic women. Parasitaemia correlated positively with IL‐10 and CXCL‐10 levels but inversely with IFN‐γ and IL‐17A. Haemoglobin levels were higher in women with low IL‐10 and CXCL‐10 levels, and in group with high IFN‐γ, IL‐17A and IL‐23 levels. Only IL‐10 levels associated negatively with parity. Positive correlations were observed between Th17 (IL‐17A) and Th1 (IFN‐γ, TNF‐α), IL‐10 family (IL‐19 and IL‐22) and Th17 (IL‐23) cytokines. Multivariate analysis showed association between: mother's age and IFN‐γ levels, parasitaemia and IL‐10 and CXCL‐10 levels and haemoglobin levels, gestational age and IL‐17A levels. In conclusion, during PAM, CXCL‐10 and IL‐10 responses are implicated in the pathogenesis while Th17 and Th1 immune responses, via IL‐17A and IFN‐γ might play protective roles.     

Role of Th1 and Th2 Cytokines in Immune Response to Uncomplicated Plasmodium falciparum Malaria

The relative balance between Th1 and Th2 cytokines appears crucial, since the role of cytokines has been evaluated in several studies by comparison of clinically heterogeneous groups of patients. The aim of this study is to determine the role of proinflammatory Th1 cytokines, interleukin-12 (IL-12) and gamma interferon (IFN-γ), and anti-inflammatory Th2 cytokines, IL-4 and IL-10, in a homogeneous group of patients with uncomplicated Plasmodium falciparum malaria. Levels of IL-12, IFN-γ, Il-4, and IL-10 in serum for 20 adult patients and 15 healthy control subjects were determined by an immunoenzymatic assay. Serum levels of Th1 cytokines, IL-12 (8.6 ± 2.8 pg/ml; controls, 3.2 ± 0.7 pg/ml) and IFN-γ (39.2 ± 67.6 pg/ml; controls, 8.4 ± 6.3 pg/ml), were significantly increased at admission; 3 days later, levels of IL-12 in serum remained significantly high (8.8 ± 2.6 pg/ml), whereas IFN-γ levels returned to control values. The anti-inflammatory response of Th2 cytokines (IL-10 and IL-4) was distinct. Levels of IL-10 in serum were not significantly increased at day 0 and day 3 (306.6 ± 200.4 pg/ml and 56.6 ± 38.4 pg/ml, respectively; controls, 17.4 ± 9.0 pg/ml). In contrast, levels of IL-4 in serum were not increased on admission (3.4 ± 1.2 pg/ml; controls, 2.4 ± 0.8 pg/ml), but at day 3 a moderate and significant increase of IL-4 levels was observed (4.5 ± 1.7 pg/ml). In conclusion, the increase of Th1 cytokine IL-12 and IFN-γ levels during the acute phase of uncomplicated P. falciparum malaria may reflect an early and effective immune response regulated by proinflammatory Th1 cytokines, and in particular IFN-γ may play a role in limiting progression from uncomplicated malaria to severe and life-threatening complications.     

Mycobacterium tuberculosis Coinfection Has No Impact on Plasmodium berghei ANKA-Induced Experimental Cerebral Malaria in C57BL/6 Mice

Cerebral malaria (CM) is the most severe complication of human infection with Plasmodium falciparum. The mechanisms predisposing to CM are still not fully understood. Proinflammatory immune responses are required for the control of blood-stage malaria infection but are also implicated in the pathogenesis of CM. A fine balance between pro- and anti-inflammatory immune responses is required for parasite clearance without the induction of host pathology. The most accepted experimental model to study human CM is Plasmodium berghei ANKA (PbANKA) infection in C57BL/6 mice that leads to the development of a complex neurological syndrome which shares many characteristics with the human disease. We applied this model to study the outcome of PbANKA infection in mice previously infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis is coendemic with malaria in large regions in the tropics, and mycobacteria have been reported to confer some degree of unspecific protection against rodent Plasmodium parasites in experimental coinfection models. We found that concomitant M. tuberculosis infection did not change the clinical course of PbANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. The immunological environments in spleen and brain did not differ between singly infected and coinfected animals; instead, the overall cytokine and T cell responses in coinfected mice were comparable to those in animals solely infected with PbANKA. Our data suggest that M. tuberculosis coinfection is not able to change the outcome of PbANKA-induced disease, most likely because the inflammatory response induced by the parasite rapidly dominates in mice previously infected with M. tuberculosis.     

Plasma Concentration of Malaria Parasite-Derived Macrophage Migration Inhibitory Factor in Uncomplicated Malaria Patients Correlates with Parasitemia and Disease Severity

Host macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of malaria infections. Several Plasmodium parasite-derived MIFs were identified to have the potential to regulate host immune response. However, the role of Plasmodium MIFs in the immunopathogenesis of malaria infection and the relationships between these mediators and inflammatory cytokines remained unclear. In this study, we have investigated two Plasmodium MIFs in peripheral blood of uncomplicated malaria patients and analyzed their correlations with several major factors during malaria infection. We found that both Plasmodium falciparum MIF (PfMIF) and Plasmodium vivax MIF (PvMIF) levels in patients were positively correlated with parasitemia, tumor necrosis factor alpha, interleukin-10 (IL-10), and monocyte chemoattractant protein 1 but were not correlated with transforming growth factor β1 and IL-12. Of interest was that the PvMIF level was positively correlated with host body temperature and human MIF (HuMIF) concentrations. Moreover, multiple stepwise regression analysis also showed that parasitemia, IL-10, and HuMIF expression were significant predictors of Plasmodium MIF production. In addition, during antimalarial drug treatment, the decreasing of Plasmodium MIF concentrations was followed by parasitemia in most patients. Our results suggested that the Plasmodium MIF circulating level reflects the level of parasitemia and thus was closely correlated with disease severity in uncomplicated malaria. Therefore, this factor has the potential to be a promising disease predictor and is applicable in clinical diagnosis.Malaria is one of the major global health problems, causing more than 300 million clinical cases and about one million deaths each year in tropic and subtropic areas. Among the four malaria parasite species that infect humans, Plasmodium falciparum and Plasmodium vivax are the most widely distributed ones, and the latter parasite is the dominating cause of malaria in southern China. Infection with P. falciparum or P. vivax provokes a complex immune response of the host, in which the cytokine network plays an essential role. One of the cytokines, macrophage migration inhibitory factor (MIF), has been considered a critical regulator in both innate and adaptive immune responses (5) and is involved in the pathogenesis of several parasitic infections (26, 32). In murine models of malaria, host MIF inhibited erythroid, multipotential, and granulocyte-macrophage progenitor-derived colony formation, and the circulating level of host MIF during Plasmodium chabaudi infection has been found to correlate with disease severity (21). Significantly, McDevitt et al. reported that the infection of MIF knockout mice with P. chabaudi was found to result in less-severe anemia, improved erythroid progenitor development, and increased survival compared to those of wild-type controls (24). In human malaria patients, MIF also was found to inhibit erythroid differentiation and hemoglobin production, further indicating that it plays a critical role in the pathogenesis of malarial anemia (24). Moreover, many reports have confirmed that MIF levels in malaria patients were altered compared to those of counterparts of noninfected individuals; however, the increase or decrease of MIF circulating levels was not consistent in these research studies, mostly due to their different sample sources and the extent of disease severity. For example, using child patient samples, Clark et al. found that children with cerebral malaria (CM) expressed very low MIF levels in blood vessel walls within the brain, whereas children with fatal P. falciparum malaria expressed high MIF levels in blood vessel walls within their peripheral tissue (9, 10). Awandare et al. reported that children with acute malaria or severe anemia showed a reduction in plasma MIF levels (3, 4). Women with malaria during pregnancy showed an increase in MIF levels in intervillous plasma and cultured intervillous blood mononuclear cells but not in the peripheral plasma (6-8). McDevitt et al. (24) and Femandes et al. (14) found an increase in plasma MIF levels in general patients with acute malaria; however, in experimental human P. falciparum malaria models, plasma MIF levels decreased significantly during early-blood-stage infection, which paralleled a similar decline in circulating lymphocytes (13). Collectively, those observations indicate that host MIF is a critical pathogenesis-associated cytokine manifested during malaria infection, and it is one that also participates in its immunopathology.In recent years, several Plasmodium MIFs have been identified and subsequently confirmed to have potential in regulating host immune responses in vitro (2, 11, 30). However, the role of Plasmodium MIFs, especially in immunopathogenesis during malaria infection, still is obscure. Cordery et al. have shown an elevation of anti-P. falciparum MIF (PfMIF) IgG levels in patients with P. falciparum (11). Our group developed a monoclonal antibody (MAb 1B9) that specifically recognized PfMIF but not host MIF and other Plasmodium MIFs, and we also identified its exact epitope in the PfMIF sequence (33). By using this MAb, we have set up a specific sandwich enzyme-linked immunosorbent assay (ELISA) method for detecting and quantifying circulating PfMIF molecules in acute malaria patients (30). In this report, we describe another monoclonal antibody (MAb 3B4) that specifically recognized P. vivax MIF (PvMIF), and a specific sandwich ELISA method was established with 3B4 for PvMIF quantification. With these MAbs we investigated the circulating PfMIF and PvMIF levels in the plasma of malaria patients from the northern part of Burma, and we analyzed the correlations with some important mediators and inflammatory factors taking place during malaria infection. Additionally, the effects of individual HLA differences on Plasmodium MIF circulating levels were determined. Finally, we determined the alterations of Plasmodium MIF concentrations during antimalarial drug treatment. Collectively, these results will help us better understand the role of Plasmodium MIF during malaria infection.     

Evidence for Multiple Pathologic and Protective Mechanisms of Murine Cerebral Malaria

Murine cerebral malaria (CM) induced by Plasmodium berghei ANKA kills susceptible mice within 24 to 48 h of onset of symptoms and is characterized by the production of inflammatory cytokines in the brain. C57BL/6J mice are sensitive to lethal CM, while A/J mice are resistant. These strains of mice were immunized with an adjuvant vaccine of killed whole-blood-stage parasites. The immunization protected C57BL/6 mice from lethal CM following virulent challenge. The same immunization increased the incidence of lethal CM in A/J mice challenged similarly. Histopathologic examination of the brains of mice from these studies revealed two distinct types of lesions. Type I CM is acute in onset; usually lethal; and characterized by widespread microglial activation, endothelial cell damage, and microvascular disruption in the brain. Type II CM is characterized by intense, but focal, mononuclear cell inflammation without endothelial cell damage or microvascular destruction. Animals with type II lesions were clinically normal and protected from type I lesions. Available clinical, epidemiological, and biochemical evidence suggests that type I and type II lesions might exist in human CM as well.     

IRGM3 Contributes to Immunopathology and Is Required for Differentiation of Antigen-Specific Effector CD8+ T Cells in Experimental Cerebral Malaria

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3^−/− but not Irgm1^−/− mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8⁺ T cells within the brain. Although antigen-specific proliferation of transferred CD8⁺ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3^−/− recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3^−/− mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3^−/− mice occurs due to impaired generation of CD8⁺ effector function. This defect is nonintrinsic to CD8⁺ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.