a-tocopherol transfer protein inhibition is effective in the prevention of cerebral malaria in mice 1 3

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1 AJCN. First published ahead of print November 18, 2009 as doi: /ajcn a-tocopherol transfer protein inhibition is effective in the prevention of cerebral malaria in mice 1 3 Maria S Herbas, Mikiko Okazaki, Eri Terao, Xuenan Xuan, Hiroyuki Arai, and Hiroshi Suzuki ABSTRACT Background: Nutritional status likely plays an important role in determining the outcome of protozoan infections. Despite the evidence of Plasmodium sensitivity to oxidative stress, the potential role of vitamin E, a free radical scavenger, on the outcome of cerebral malaria (CM) has yet to be elucidated. Objective: To determine the influence of vitamin E on Plasmodium parasite development and murine CM outcome, a-tocopherol transfer protein (a-ttp), a regulator of vitamin E in the host circulation, was abrogated. Design: a-ttp knockout mice were infected with P. berghei ANKA, and survival rate, parasitemia, brain histologic alterations, and brain barrier permeability were assessed. In addition, mrna expression of the cytokines and adhesion molecules involved in this neurologic pathology were monitored. Results: a-ttp knockout mice infected with P. berghei ANKA did not exhibit any clinical or pathologic signs of CM, and a histologic analysis of the brain tissues in these animals showed no alteration of blood-brain barrier integrity compared with that in control mice. Interestingly, protection of the blood-brain barrier in these infected a-ttp knockout mice was lost when dietary supplementation with vitamin E was added to their diet. Moreover, interleukins and adhesion molecule transcripts in the brain of control mice were significantly up-regulated compared with those in the a-ttp knockout mice. Conclusion: It appears that a deficiency of a-tocopherol in the circulation prevents CM and suggests that a-ttp is a putative target for the early prevention of CM. Am J Clin Nutr doi: /ajcn INTRODUCTION Malaria is widespread throughout tropical and subtropical regions, causing.300 million acute illnesses and resulting in at least one million deaths annually (1). The presence of Plasmodium falciparum in humans results in red blood cell infection and diminution (malarial anemia), with a consequent clogging of capillaries that carry blood to the brain (cerebral malaria) as well as other organs (2). Several factors are likely involved in disease pathogenesis, such as human genetics, malaria parasite genetics, nutritional status of the host, and concurrent infections (3). In humans, cerebral malaria is initiated by sequestered parasitized erythrocytes in blood vessels that elicit a host response, which includes local or systemic cytokine production, the presence of both CD4 + and CD8 + T cells (2, 4), and alterations in the pattern of release of neuroactive mediators (4). In addition, histopathologic lesions stemming from focal intravascular sequestration of mononuclear cells and parasitized erythrocytes are typically evident (5). The current antimalarial treatment is focused on killing the erythrocytic form of the parasite, and, as such, there is currently no specific treatment of cerebral malaria. Thus, the identification of new drug targets in the host would be a critical step in the prevention, treatment, and control of this cerebral pathology. Mouse models for cerebral malaria constitute an important tool for such a step, because they closely mimic the features of human cerebral malaria (6), including the intravascular sequestration of macrophages and monocytes and the breakdown of blood-brain barrier permeability (5, 7). Research on the oxidative stress status of the host before and during infection has emerged as a subject of intense interest, resulting in the finding that Plasmodium parasites are sensitive to oxidative stress (8, 9). Furthermore, deficiencies of intra- and extracellular free radical scavengers, such as vitamin E, seem to make Plasmodium parasites more susceptible to oxidative attack (8, 10, 11). Vitamin E deficiency might also inhibit the development of cerebral malaria, although the mechanism by which this phenomenon occurs is not clear (12). However, dietary manipulation is an unviable strategy because of the high efficiency of vitamin E salvaged from the liver into the circulation by a-tocopherol transfer protein (a-ttp). a-ttp is a cytosolic protein in the liver responsible for the vitamin E concentration in the host circulation (13) and for facilitating its release for distribution to peripheral tissues (14). We have used a-ttp knockout mice (15) to investigate the effect of vitamin E deficiency on the development of murine cerebral malaria and found host resistance to be reversible by dietary supplementation with vitamin E. 1 From the Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan (MSH, MO, ET, XX, and HS), and the Graduate School of Pharmaceutical Science, The University of Tokyo, Tokyo, Japan (HA). 2 Supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 3 Address correspondence and requests for reprints to H Suzuki, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido , Japan. hisuzuki@obihiro.ac.jp. Received June 19, Accepted for publication October 21, doi: /ajcn Am J Clin Nutr doi: /ajcn Printed in USA. Ó 2009 American Society for Nutrition 1 of 8 Copyright (C) 2009 by the American Society for Nutrition

2 2of8 HERBAS ET AL MATERIALS AND METHODS Animals and experimental infections Inhibition of a-ttp has been shown to lead to undetectable concentrations of vitamin E in the circulation (15). Adult a-ttp knockout mice with a C57BL/6J genetic background (15) and C57BL/6J control mice were housed in polycarbonate cages and maintained as specific pathogen free animals in a light-controlled (lights on from 0500 to 1900) and air-conditioned room of the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine. The animalroom temperature was set at C, and humidity was set at %. Mice had free access to a standard laboratory nonpurified diet containing 75 mg vitamin E/kg (CE-2; CLEA Japan, Tokyo, Japan). At 8 wk of age, 6 mice in each experimental group were infected intraperitoneally with infected red blood cells (IRBCs) of P. berghei ANKA. Parasitemia was monitored every other day by examining Giemsa-stained blood smears collected from the mice tail vein. Survival rate and pathologic symptoms, such as paralysis, convulsions, stupor, and rolling over, were monitored daily. In addition, hematologic variables such as the hemoglobin concentration (g/dl), hematocrit percentage (%), and RBC number were recorded throughout the course of infection. Briefly, 5 ll blood taken from the tail vein was mixed in an isotonic buffer (Isotonac, MEK-510; Nihon Kohden, Tokyo, Japan) and analyzed with an automatic hematological analyzer (Celltac a, MEK-6358; Nihon Kohden). All of the animal experiments described herein were conducted in accordance with the standards relating to the Care and Management of Experimental Animals of Obihiro University of Agriculture and Veterinary Medicine, Japan. Vitamin E treatment To increase a-tocopherol concentrations in the circulation, 10 days before and throughout infection the a-ttp knockout (n = 6) and control C57BL/6J (n = 6) mice were fed the standard diet to which a-tocopherol had been added to a total concentration of 600 mg/kg (CLEA Japan). When a-ttp homozygous mutant mice were fed vitamin E supplemented diets, plasma a-tocopherol concentrations were maintained within the normal range, close to the concentrations in heterozygous mutants fed a normal diet (15). Then, mice were infected with IRBCs of P. berghei ANKA. Signs and symptoms of cerebral malaria, parasitemia, and survival rate were monitored as described above. The experimental design for both of the infectious experiments is shown in Figure 1. Evaluation of blood-brain barrier permeability On days 0, 5, 7, 15, and 25 after infection with P. berghei ANKA, a-ttp knockout (n = 4) and C57BL/6J (n = 4) mice were injected with 0.2 ml of 1% Evans blue solution into the tail vein intravenously, and 2 h later the mice were killed and their brains removed. Blood-brain barrier permeability was evaluated by the previous Evans blue staining of brain tissues. Additionally, brain-barrier permeability was also evaluated in mice fed a vitamin E supplemented diet following the same protocol as described above. Tissue collection and histology On days 0, 5, 7, 8, 15, 20, and 25 after infection, a-ttp knockout (n = 4) and C57BL/6J (n = 4) mice infected with IRBCs were anesthetized with diethyl ether (Wako, Tokyo, FIGURE 1. Experimental design for the infectious experiments. A: In the first experiment, a-tocopherol transfer protein knockout mice fed a normal diet (a-ttp D ) (75 mg/kg) were infected with Plasmodium berghei ANKA, after which parasitemia levels and survival rates were monitored. Brains were removed at different time points after infection, and Evans blue assay (sampling days: 0, 7, 15, and 25), histologic analysis (sampling days: 0, 7 or 8, 15, and 25), and mrna expression of cytokines and adhesion molecules (sampling days: 0, 7, 15, and 20) were performed. B: In a second experiment, mice were fed a vitamin E supplemented diet (600 mg/kg) from 10 d before infection, after which levels of parasitemia and survival rates were monitored. Brains were removed, and Evans blue assay was performed on days 0, 7, 15, and 25 after infection. IRBCs, infected red blood cells.

3 VITAMIN E DEFICIENCY AND CEREBRAL MALARIA 3 of 8 Japan). Blood was collected by cardiac puncture, after which the mice were killed by cervical dislocation, and the brain, liver, and spleen were aseptically removed. The liver and spleen were immediately put in liquid nitrogen and stored at 280 C. The brain was maintained in 10% formalin for 24 h. Thereafter, tissues were treated with ethanol at concentrations of 70%, 80%, 90%, 95%, and 100% (Wako) and then with xylene 3 times at a concentration of 100% (Wako). The tissues were then embedded in paraffin and cut into sections of 5-lm thickness. Subsequently, sections were stained with hematoxylin-eosin, mounted, and analyzed under light microscopy. Gene expression analysis by real-time quantitative polymerase chain reaction Genes involved in the inflammatory and antiinflammatory responses, such as interferon (IFN)-c, tumor necrosis factor (TNF)-a, interleukin (IL)-10, and IL-1b as well as genes involved in the adhesion process, such as vascular cellular adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), lymphocyte function associated antigen (LFA-1), and glial fibrillary acidic protein (GFAP) were analyzed as target genes. Total RNA from the brain, spleen, and liver of uninfected (n = 6) and infected (n = 6) mice on days 0, 7, 15, and 20 was extracted by using Trizol (Sigma, St Louis, MO). mrna was purified by using a Poly purist extraction kit (Ambion, Austin, TX), and the concentration was adjusted to 100 ng/ll before serial dilution. RNA quality was assessed by using Experion automatized chromatography (Experion TM RNA StdSens Analysis Kit; Bio-Rad, Hercules, CA). Real-time quantitative polymerase chain reaction (PCR) was performed with specific doubly labeled probes in an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, the reaction mixture contained 10 ll of 2 master mix without UNG (uracil-n-glycosylase), 0.5 ll of 40 multiscribe and RNase inhibitor mix, 4 ll of 50 ng/ll RNA template, 1.1 ll RNase free double-distilled water, and 1 ll TaqMan gene expression assay probe. The reaction program was as follows: 40 C for 30 min, 95 C for 10 min, 45 cycles with 95 C for 15 sec, and 60 C for 1 min. The data were analyzed by using SDS 2.1 software (Applied Biosystems). Mouse b-actin, 18SrRNA, and GAPDH were used as internal control genes. Genorm_win_3.5 software (16) was used for the selection of the most stable internal control gene. For a description of the primers and probes used for the internal control genes, see Supplementary Table 1 under Supplemental data in the online issue; for a description of the primers and probes used for the target genes, see Supplementary Table 2 under Supplemental data in the online issue. All of the experiments described here were repeated 3 times. Statistical analysis Statistical analysis was performed by using one-factor and 2-factor analysis of variance. Multiple comparisons were performed by using the Scheffe multiple comparison test (Stats- Direct, version 2.7.5, software for Windows). The Kaplan-Meier method was used for the survival rate analysis. A P value,0.05 was considered statistically significant. RESULTS Experimental infection and hematologic measurements After infection with P. berghei ANKA, control mice exhibited neurologic symptoms, including convulsions, paralysis of the limbs, and rolling over before the onset of death on day 7 after FIGURE 2. a-tocopherol transfer protein (a-ttp) knockout mice fed a normal diet (a-ttp D ) showed reduced Plasmodium berghei ANKA development and malaria progression. Parasitemia kinetics (A), survival rates (B), and gametocyte percentages (C) in a-ttp D mice (n = 6) and C57BL/6J control mice (n = 6). A and C: *P, 0.05 for C57BL/6J compared with a-ttp D mice on days 7 and 8, respectively. A: **P, 0.05 for day 0 compared with day 7 for C57BL/6J mice. B: *P, 0.01 for C57BL/6J compared with a-ttp D mice. Arrows indicate 100% death of the animals.

4 4of8 HERBAS ET AL FIGURE 3. Vitamin E supplementation triggers the development of cerebral malaria. Parasitemia kinetics (A) and survival rates (B) in a-tocopherol transfer protein (a-ttp) knockout mice fed a normal diet (a-ttp D ), a-ttp knockout mice fed a vitamin E supplemented diet (a-ttp D E), and control C57BL/6J mice fed a normal diet (B6) or a vitamin E supplemented diet (B6E). n = 6 per group. A: *P, 0.05 for B6 (75 mg/kg), B6E (600 mg/kg), and a-ttp D E (600 mg/kg) compared with a-ttp D (75 mg/kg) on day 6; **P, 0.05 for a-ttp D (75 mg/kg) compared with a-ttp D E (600 mg/kg) on day 8; ***P, 0.05 for B6E (600 mg/kg) compared with a-ttp D E(600mg/kg)onday8.B:*P, 0.01 for B6 (75 mg/kg), B6E (600 mg/kg), and a-ttp D E compared with a-ttp D. Arrows indicate 100% death of the animals. infection. Strikingly, none of these symptoms were observed in a-ttp knockout mice, which suggests the strong involvement of this gene in Plasmodium development. By day 7 after infection, a-ttp knockout and control mice exhibited 0.2 1% and 10 14% parasitemia, respectively (Figure 2A). The hemoglobin concentration was g/dl and g/dl in C57BL/6J and a-ttp knockout mice, respectively. Whereas control mice died on day 7 after infection, a-ttp knockout mice required a further 8 d to reach parasitemia levels matching those of control mice at day 7 and survived for 27 to 30 d (Figure 2B). Furthermore, these knockout mice failed to exhibit symptoms of cerebral malaria despite reaching parasitemia levels between 50% and 60% and eventually succumbed when displaying signs of severe anemia; the hemoglobin concentration was g/dl FIGURE 4. Blood-brain barrier permeability damage after vitamin E supplementation. Blood-brain barrier permeability was evaluated by using Evans Blue stain an indicator of capillary permeability in a-tocopherol transfer protein (a-ttp) knockout mice fed a normal diet (75 mg/kg) (a-ttp D ), a-ttp knockout mice fed a vitamin E supplemented diet (a-ttp D E), and control C57BL/6J mice fed a normal diet. a-ttp D mice did not exhibit any leakage throughout the infection (D, E, and F). In contrast, a positive reaction was detected in C57BL/6J mice (C). Uninfected mice are represented in panels A, B, and G. Evans blue stain was positive for a-ttp D E mice (H). Vitamin E restoration in the host circulation induces the development of CM. n =4.

5 VITAMIN E DEFICIENCY AND CEREBRAL MALARIA 5 of 8 FIGURE 5. Histologic analysis of brains. Brain sections were stained with hematoxilin-eosin stain, and histologic alterations such as disruption of the vessel wall, enhancement of the perivascular space, and intravascular accumulation of mononuclear cells were observed in C57BL/6J mice (B and C), whereas such alterations were not detected in brains from a-tocopherol transfer protein (a-ttp) knockout mice fed a normal diet (a-ttp D ; E and F). Uninfected mice are represented in panels A and D. a-ttp D mice did not exhibit histologic alterations on days 15 and 25 after infection (G and H), even at the same parasitemia levels at which control C57BL/6J mice developed cerebral malaria. n = 4. Arrows indicate 100% death of the animals. on day 20 after infection. The gametocyte percentage in a-ttp knockout mice was significantly lower during the acute phase of infection (Figure 2C). Taken together, it appears that a-ttp plays a critical role in Plasmodium development and the onset of cerebral malaria. Vitamin E treatment Whereas parasitemia remained significantly lower in the a-ttp knockout mice fed a normal diet than in all of the other groups (P, 0.05), the a-ttp knockout mice fed a diet supplemented with vitamin E had parasitemia kinetics similar to those of control C57BL/6J mice fed either a normal diet or a diet supplemented with vitamin E (Figure 3A). Importantly, whereas a-ttp knockout mice fed a normal diet survived for 30 d and failed to exhibit symptoms of cerebral malaria, the same mice supplemented with vitamin E died after only 8 10 d in a manner similar to that of control mice (Figure 3B). Furthermore, the a-ttp vitamin E supplemented mice exhibited neurologic symptoms of cerebral malaria, including convulsions, paralysis of the limbs, and rolling over before the onset of death. These results strongly suggest that a-ttp is exerting its effects on Plasmodium and disease development indirectly through vitamin E concentrations in the host. Evaluation of blood-brain barrier permeability The breakdown of the blood-brain barrier can be detected by using an Evans blue assay that stains brains in which the barrier has been lost. Because the control mice infected with P. berghei ANKA displayed the symptoms of cerebral malaria, it was expected that disease progression was due to breakdown of the blood-brain barrier; the staining in these mice confirmed this suspicion (Figure 4C). In contrast, the brains of a-ttp knockout mice failed to show any brain capillary leakage throughout the course of the infection (Figure 4, D, E, and F). Tellingly, the brains of a-ttp knockout mice fed a vitamin E supplemented diet, however, showed blood-brain barrier breakdown (Figure 4H). This breakdown of the blood-brain barrier matches the disease progression kinetics seen in all of the groups. Brain tissue histology Brain sections from C57BL/6J control mice displayed severe lesions, such as hemorrhage, disruption of the vessel wall,

6 6of8 HERBAS ET AL FIGURE 6. Mean (6SEM) mrna expression of interferon-c (IFN-c), tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and interleukin-10 (IL-10) in the brains of a-tocopherol transfer protein (a-ttp) knockout mice fed a normal diet (a-ttp D ) and in control C57BL/6J mice fed a normal diet. Significant up-regulation of cytokines in the brain of C57BL/6J mice was observed before the onset of death. Cytokine expression in the brains of a-ttp D mice remained stable. n = 6. Bars with different lowercase letters are significantly different, P, 0.05 (Scheffe multiple-comparisons test). enhancement of the perivascular space, and intravascular accumulation of mononuclear cells, before the onset of death. These lesions were located mainly in the cerebellum and cerebrum (Figure 5, B and C). In contrast, the brain sections of the a-ttp knockout mice displayed no vascular abnormalities throughout the course of infection (Figures 5, E and F), despite parasitemia in these mice reaching levels that were sufficient for the development of cerebral malaria in susceptible mice (Figures 5, G, H, and I). Analysis of mrna cytokine expression in the brain by real-time quantitative PCR The expression levels of all the cytokines tested were significantly higher in the brains of C57BL/6J mice after infection (P, 0.05) (Figure 6). Importantly, the transcript levels of these cytokines was significantly greater in the brains of C57BL/6J mice than of a-ttp knockout mice on day 7 after infection (P, 0.05) (Figure 6), which suggests that control mice were mounting an immune response. Surprisingly, despite the lack of symptoms of cerebral malaria, mrna expression levels of all the cytokines tested were significantly up-regulated in the brains of the a-ttp knockouts from day 15 onward after infection. However, aside from IL-1b, cytokine expression remained lower than in control mice (Figure 6). Adhesion molecules are up-regulated in the brains of infected animals On day 6, during the height of acute infection, mrna expression levels of ICAM, LFA-1, and gliar fibrillary acidic FIGURE 7. Mean (6SEM) mrna expression of vascular cellular adhesion molecule (VCAM), intracellular adhesion molecule (ICAM), lymphocyte function associated antigen (LFA-1), and glial fibrillary acidic protein (GAFP) in the brains of a-tocopherol transfer protein (a-ttp) knockout mice fed a normal diet (a-ttp D ) and in control C57BL/6J mice fed a normal diet. Significant up-regulation of adhesion molecules in brains of C57BL/6J mice before onset of death was observed. Expression levels in the brains of a-ttp D mice remained stable throughout the course of infection. n = 6. Bars with different lowercase letters are significantly different, P, 0.05 (Scheffe multiple-comparisons test). protein (GAFP) were all significantly up-regulated in control mice, in contrast with the a-ttp knockout mice, in which the expression of ICAM and LFA-1 was significantly up-regulated from day 15 after infection (Figure 7). In addition, mrna expression levels of GAFP remained unaffected in the a-ttp knockout mice. Surprisingly, VCAM expression was unaffected during infection, which suggests that this adhesion molecule may not play a role in this process in mice. Overall though, it appears that there is a transcriptional up-regulation of adhesion molecules known to play a role in RBCs and mononuclear cell sequestration in the control animals a change that is not reflected in their knockout counterparts, which suggests that this process does not occur in the a-ttp knockout mice. Analysis of mrna IFN-c and TNF-a gene expression in the liver and spleen by real-time quantitative PCR The levels of cytokine expression in both the a-ttp knockout and the C57BL/6J control mice were increased in the liver and spleen (Figure 8). Transcription appeared significantly higher in the control mice than in the knockout mice during the acute phase of infection, which likely reflected an increased parasite load in these animals. Taken together, it appears that immunity was normal in these knockout mice, which suggests that protection is not a result of a compromised immune response in these animals.

7 VITAMIN E DEFICIENCY AND CEREBRAL MALARIA 7 of 8 FIGURE 8. Mean (6SEM) interferon-c (IFN-c) and tumor necrosis factor-a (TNF-a) expression levels in the livers and spleens of a-tocopherol transfer protein (a-ttp) knockout mice fed a normal diet (a-ttp D ) and in control C57BL/6J mice fed a normal diet. Increased expression levels of the inflammatory cytokines were observed after infection in both genotypes, which indicated a normal immune response of the host against infection. n = 6. Statistical comparisons were made with a Scheffe multiple-comparisons test. A, B, and D: *P, 0.01 for C57BL/6J compared with a-ttp D on day 7; **P, 0.01 for day 0 compared with day 7 for both genotypes; ***P, 0.01 for day 0 compared with day 6 for both genotypes. C: ****P, 0.01 for day 6 compared with day 7 for both genotypes. A and D: *****P, 0.01 for day 0 compared with day 15 for a-ttp D. DISCUSSION Cerebral malaria is a neuroinfectious disease, the pathologic mechanisms of which remain unclear. However, it was reported previously that dietary-induced vitamin E deficiency in mice was protective against the development of this disease (12). Exploitation of this putative treatment, however, would be an impractical strategy because of the content of vitamin E in daily food. Thus, a transient inhibition of a-ttp activity in the liver would be useful and would also minimize any side effects that diet-induced vitamin E deficiency might trigger. We showed that anemia was absent in a-ttp knockout mice, in contrast with C57BL/6J control mice fed a vitamin E deficient diet (17). Clues to this anemia avoidance can be found in patients with familial isolated vitamin E deficiency, who possess normal mechanisms for vitamin E absorption (18). It appears that a-ttp knockout mice also absorb vitamin E from a normal diet and maintain transiently acceptable concentrations of vitamin E in RBC membranes and other tissues, thereby avoiding anemia (19). The a-tocopherol transfer protein belongs to the family of lipid binding proteins characterized by the Sec14 domain and contains a lipid binding pocket site, where a-tocopherol binds (13), making it a potential inhibitory drug target site (13, 20). The outcome of cerebral malaria is determined during the acute phase of the infection when parasitemia reaches values between 10% and 15%. At this stage, mice display characteristic signs, such as paralysis of the limbs, ataxia, deviation of the head, spontaneous rolling over, convulsions, and coma (1). In this study, P. berghei ANKA infected wild-type mice died showing typical signs of cerebral malaria. This finding contrasts with that in a-ttp knockout mice, which did not exhibit these signs throughout the course of infection, despite parasitemia levels rising above those seen in wild-type mice close to death (Figure 2). In addition, brain sections from a-ttp knockout mice did not exhibit any histologic alterations at the parasitemia levels at which severe brain damage was observed in C57BL/6J mice (Figure 5). Parasites sequestered in erythrocytes can elicit an immune response that includes cytokine production in humans (1). RBC sequestration has been observed in mice, although the exacerbated immune response is attributed to mononuclear cell sequestration (1). The brains of a-ttp knockout and control C57BL/6J mice infected with P. berghei ANKA were therefore removed to monitor the transcript expression of cytokines such as IFN-c, TNF-a, IL-1b, and IL-10. In the brains of a-ttp knockout mice, these molecules were not increased during the acute phase of infection (Figure 6). However, mrna expression levels of TNF-a and IFN-c in the liver and spleen were strongly up-regulated for both genotypes, which indicated that a-ttp knockout mice possess a normal immune response against infection (Figure 8). Attempts by the host to mount a defense response during Plasmodium infection include sequestration of the parasitized RBCs and intravascular accumulation of mononuclear cells, a process mediated by increased transcription of several endothelial cell mediators, including ICAM-1 (21, 22). Therefore, mrna expression levels of various adhesion molecules (ICAM, VCAM, LFA-1, and GAFP) in the brains of a-ttp knockout and control mice infected with P. berghei ANKA were examined. The expression of ICAM, LFA-1, and glial

8 8of8 HERBAS ET AL fibrillary acidic protein were significantly increased after the infection in C57BL/6J mice. In contrast, the expression of these molecules in a-ttp knockout mice was not increased during the acute phase of the infection (Figure 7). Interestingly, ICAM-1 deficient mice (ICAM-1 is involved in the sequestration of infected RBCs) infected with P. berghei ANKA also failed to develop cerebral malaria, even though parasitemia levels were similar to those observed in susceptible wild-type mice (7). It is thus unlikely that the parasitemia level by itself has critical influence on the outcome of cerebral malaria. The other factors influencing the onset and outcome of cerebral malaria remain to be determined. Considering that a-ttp knockout mice died without displaying any symptoms of cerebral malaria, either superficially or histologically, death of the mice likely occurred from other causes. Anemia seems to be a likely cause. Interestingly, it is not just a-ttp knockout mice that succumb to anemia, because it was reported previously that a significant proportion of wild-type mice that overcame the acute phase of cerebral malaria eventually died of anemia (12). In the present study, 25% of wild-type mice that overcame the acute phase of cerebral malaria and might have died from anemia, although death occurred sooner in these mice than in the a-ttp knockout mice. Another key concern raised in this study was the cause of the inhibition of parasite proliferation, not only in the a-ttp knockout mice but also in the wild-type mice deprived of vitamin E. A previous study showed that the protective effects of vitamin E deficiency were attributed to the effects on the pro-oxidant status of the host that had inhibited growth of the parasite (12). Moreover, Levander et al (23) suggested that inhibition of parasite proliferation was due to parasite membrane cellular damage. Absence of the free radical scavenger properties of vitamin E is likely to trigger an imbalance in the redox status of infected RBCs, which would in turn inhibit the proliferation of parasites during the erythrocytic cycle. Consistent with this idea, we observed that DNA from parasites infecting knockout mice was damaged oxidatively (M Herbas and H Suzuki, unpublished observations, 2006). In addition, the significantly lower percentage of gametocytes observed during the first peak of parasitemia in knockout mice suggests that the normal erythrocytic cycle of these parasites was delayed during the acute phase of the infection (Figure 2C), possibly as a result of parasite DNA damage. Because CD36 is known to be a receptor of Plasmodium and is down-regulated by vitamin E (24), additional study is needed to clarify the possible role of CD36 regulation by vitamin E in the observed protective effect against cerebral malaria. 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