The liver stage of malaria, caused by the genus Plasmodium, is clinically silent, but immunologically significant. Ample evidence exists for an effective CD8+ T cell response to this stage as well as the involvement of γδT cells and NK1.1int cells in immunized animal models. In contrast, there is little information concerning responses in a naive host. Here we report that several host gene expressions in the liver, spleen, and kidney of BALB/c mice are altered during the liver stage of Plasmodium yoelii infection. Really interesting new gene 3 (Ring3), semaphorin subclass 4 member G, glutamylcysteine synthetase, and p45 NF erythroid 2 were all up-regulated 24 h after infection with P. yoelii. Semaphorin subclass 4 member G expression was elevated in the kidney, whereas Ring3 was elevated in both spleen and kidney. The expression of TNF-α (TNF-α and IFN-γ) were down-regulated in all three tissues tested except in infected spleen where IFN-γ was elevated. P. yoelii-related host gene changes were compared with those in Toxoplasma gondii-infected livers. Ring3 expression increased 5-fold over control values, whereas expression of the other transcripts remained unchanged. TNF-α and IFN-γ expressions were increased in the Toxoplasma-infected livers. The uniform increase of Ring3 expression in both Plasmodium- and Toxoplasma-infected livers suggests an innate immune response against parasitic infections, whereas the other gene expression changes are consistent with Plasmodium parasite-specific responses. Taken together, these changes suggest the immune responses to P. yoelii infection are both parasite and organ specific.

Despite earlier successes in reducing human malaria, the disease is now rampant in many tropical and subtropical countries, taking a heavy toll of human life. Several reasons account for the resurgence of malaria. These include parasite resistance to cheap and highly effective anti-malaria drugs and the development of mosquito vectors resistant to effective insecticides. In addition, the complexity of the life cycle of the parasite has contributed to an inability to control this disease. Thus, a multidisciplinary approach is required that targets various developmental stages of the parasite. Recent advances in parasite biology and vaccine design have created an atmosphere of hope that, within the next decade, new generation vaccines will bring malaria under control. The crucial component for a successful approach to controlling malaria is to understand host immune responses to the parasite.

Host immune responses pertaining to the erythrocytic stage have been widely studied and are believed to be primarily humoral by nature because mature erythrocytes do not express appreciable levels of the MHC. Immunologically naive children can be temporarily protected from infection by the administration of immune sera obtained from adults who were repeatedly exposed to Plasmodium falciparum (1). These Abs, which specifically target erythrocytic stage Ags, are believed to inhibit further cell invasion and cytoadherence between the infected and the uninfected erythrocytes, and mediate Ab-dependent cytotoxic activity. When compared with the erythrocytic infection, relatively less is known about the host responses that occur during the liver stage of the infection.

Although a significant amount of work has been done to assess the immune response at the liver stage to irradiated sporozoites and subunit vaccines (2, 3), much less effort has been spent studying early immune responses in the naive hosts.

Several studies have looked at cellular infiltrates such as the role of γδ T cells (4) and Kupffer cells. Indirect evidence suggests that Kupffer cells have the capacity to eliminate sporozoites. The relative number of liver stage parasites rose significantly when Kupffer cells were eliminated in vivo (5). But, little information exists about the molecular changes that may occur in the liver in response to infection.

As part of a project to identify plasmodial genes that were expressed during the liver stage, differential display analysis was undertaken on P. yoelii-infected livers. In addition to the identification of parasite genes that were expressed during the liver stage (6), mouse gene sequences were also amplified. Herein, we report the expression of several host transcripts, namely really interesting new gene 3 (Ring3),3 semaphorin subclass 4 member G (sema 4g), p45 NF erythroid 2 (p45 NF E2), glutamylcysteine synthetase (GCS), IFN-γ, and TNF-α. Furthermore, their expression appears to be regulated in a tissue- and parasite-specific manner during the liver stage of P. yoelii infection.

Female BALB/c mice, 6–8 wk old, were purchased from Charles River Breeding Laboratories (Wilmington, MA). The animals were cared for and used strictly in accordance with the University of Maryland, Baltimore Institutional Animal Care and Use Committee as well as the Public Health Services guidelines (Committee on Care and Use of Laboratory Animals, National Institutes of Health, Bethesda, MD).

Anopheles stephensi mosquitoes were fed on gametocytemic mice and maintained thereafter at 24°C and 75% humidity for 14 days. The infected mosquitoes were sugar fed, and their parasite burden was monitored by examining five mosquito midguts for oocysts. Sporozoites were isolated from these mosquitoes as previously described (6, 7). A sporozoite dose-response RT-PCR was conducted to determine the optimum sporozoite dose to detect parasite-specific stage-specific mRNA (8). Sporozoites were then resuspended in 500 μl of M199/5% FCS and injected via tail vein at a concentration of 2 × 106 sporozoites per animal. Sham-infected animals were injected with material isolated from an equal number of uninfected mosquitoes.

Sham-infected (UL) and P. yoelii-infected (IL) livers were harvested from the mice 24 h post P. yoelii infection. The livers were processed for the extraction of total RNA using TRIzol reagent (Life Technologies, Gaithersburg, MD). Two micrograms of total RNA from sham-infected and infected livers were used in the DD reaction as previously described (6). Gene-specific primers were synthesized based on the sequences of the DD clones. Table I shows the mouse transcripts, their gene-specific primer sequences, and the corresponding annealing temperatures in the PCR step. PCR began with an initial denaturing step of 94°C for 3 min followed by 94°C for 30 s, “x”°C (see Table I) for each individual transcript for 45 s; 72°C for 30 s for a total of 30 cycles, and a final extension step of 72°C for 10 min.

Table I.

Gene-specific primers and their annealing temperatures (“x”) that correspond to each host transcript

TranscriptsPrimer Sequences (forward and reverse) (5′ to 3′)Annealing Temperatures
Ring3 CCC ACA ATG GCT TCT GTA CCA G 60°C 
 CCC TGG AGT GTT GCT AAC TTG G  
GCS GCC TGC GAA AAA AGT GCC CG 60°C 
 TTC CCC TGC TCT TCA CGA TGA CCG  
Sema 4g AAC AGA CAG AGA CCT GGA ACT TGC 55°C 
 TCA TAG CGA CTG CCT AAG TGG G  
p45 NF E2 AAC TAA AAC AAA GCA AAC CCC CCG 55°C 
 CCT GCC TAC CAC AAT GAG CAA TGC  
IFN-γ AAG TTC TGG GCT TCT CCT CCT GCG 60°C 
 CGA ATC AGC AGC GAC TCC TTT TCC  
TNF-α TCC CAG AAA AGC AAG CAG CCA AC 62°C 
 CGC TTA CAG TTC CTC TTT GCC CCA C  
β-actin TAT GGA GAA GAT TTG GCA CC 55°C 
 TCA TCG TAC TCC TGC TTG C  
TranscriptsPrimer Sequences (forward and reverse) (5′ to 3′)Annealing Temperatures
Ring3 CCC ACA ATG GCT TCT GTA CCA G 60°C 
 CCC TGG AGT GTT GCT AAC TTG G  
GCS GCC TGC GAA AAA AGT GCC CG 60°C 
 TTC CCC TGC TCT TCA CGA TGA CCG  
Sema 4g AAC AGA CAG AGA CCT GGA ACT TGC 55°C 
 TCA TAG CGA CTG CCT AAG TGG G  
p45 NF E2 AAC TAA AAC AAA GCA AAC CCC CCG 55°C 
 CCT GCC TAC CAC AAT GAG CAA TGC  
IFN-γ AAG TTC TGG GCT TCT CCT CCT GCG 60°C 
 CGA ATC AGC AGC GAC TCC TTT TCC  
TNF-α TCC CAG AAA AGC AAG CAG CCA AC 62°C 
 CGC TTA CAG TTC CTC TTT GCC CCA C  
β-actin TAT GGA GAA GAT TTG GCA CC 55°C 
 TCA TCG TAC TCC TGC TTG C  

Two micrograms of total RNA was used from the UL treatment group to set up a RT using random hexamers 50 ng/μl and 200 U of Superscript II RTase (Life Technologies). PCR was conducted with the gene-specific primers (shown in Table I) and [32P]deoxyribocytidine (Amersham Pharmacia Biotech, Piscataway, NJ) at a concentration of 0.1 μCi/μl. A 5-μl sample of each reaction was removed after every five PCR cycles. These resulting PCR products were electrophoresed on a 10% Tris-buffered EDTA polyacrylamide gel, and the exponential portion of the amplification curve was plotted for each transcript. Briefly, the total counts from each amplification were measured with a phosphoimager (Molecular Dynamics, Sunnyvale, CA), and the values that corresponded to half the maximal amplification were determined graphically. Fig. 1 shows an example of the PCR exponential curve of several host gene transcripts plotted to determine the cycle number under set conditions that would correspond to the 50th percentile of the amplification. Quantitative RT-PCR was subsequently set up using 2 μg of total RNA from UL and IL groups. cDNA was then pooled into a PCR master mix, which was then aliquoted and amplified with gene-specific primers for a predetermined number of cycles (Table II). Individual transcript values were normalized to those of β-actin for both UL and IL groups, and intra-assay variation was controlled by expressing values as a percentage (%) of the control UL values.

FIGURE 1.

PCR exponential curves of β-actin, sema 4g, and TNF-α in Plasmodium yoelii-infected liver are shown in A. B, Actual phosphoimages of the PCR amplicons; C represents three of the seven transcripts investigated, and their percent maximum of amplification values are shown. Briefly, amplification was conducted for each transcript and β-actin in the same sample until the reaction plateau was reached. Total amount of amplicon per cycle was then normalized (divided by that of β-actin) to obtain a relative value. The value that was the highest due to plateau of the amplification reaction was then set at 100%, and the percent maximum values for each cycle titrated were calculated accordingly from the equation.

FIGURE 1.

PCR exponential curves of β-actin, sema 4g, and TNF-α in Plasmodium yoelii-infected liver are shown in A. B, Actual phosphoimages of the PCR amplicons; C represents three of the seven transcripts investigated, and their percent maximum of amplification values are shown. Briefly, amplification was conducted for each transcript and β-actin in the same sample until the reaction plateau was reached. Total amount of amplicon per cycle was then normalized (divided by that of β-actin) to obtain a relative value. The value that was the highest due to plateau of the amplification reaction was then set at 100%, and the percent maximum values for each cycle titrated were calculated accordingly from the equation.

Close modal
Table II.

Number of PCR cycles that corresponds to 50th percentile of the amplification curve for the seven host gene products

TranscriptsPCR Cycle No.
LiverSpleenKidney
Ring3 28 36 36 
GCS 28 NDa ND 
Sema 4g 25 ND 33 
p45 NF E2 28 ND ND 
IFN-γ 33 18 ND 
TNF-α 35 28 28 
β-actin 23 28 18 
TranscriptsPCR Cycle No.
LiverSpleenKidney
Ring3 28 36 36 
GCS 28 NDa ND 
Sema 4g 25 ND 33 
p45 NF E2 28 ND ND 
IFN-γ 33 18 ND 
TNF-α 35 28 28 
β-actin 23 28 18 
a

ND, Not detected.

The ME 49 strain of T. gondii was passaged as cysts in BALB/c mice. Twenty cysts of ME 49 strain were then injected i.p. per animal. Livers were harvested 24 h postinfection, and total RNA was extracted as described above.

Results were expressed as the mean ± SEM with n = 6 for all the Plasmodium infections. Student’s t test was used to compare differences between control and treatment groups, and the differences were assessed by a one-way ANOVA. The level of significance was set at p = 0.05.

To investigate changes in gene expression in P. yoelii-infected livers, DD was performed on UL and IL cDNA. Many DD bands were identified as being exclusive to the IL samples. Several of the DD bands were determined to be parasite gene products in the P. yoelii-infected livers (6). Four additional DD bands that were excised from the gel, reamplified, subcloned, and sequenced were identified as murine gene products. BLAST search confirmed that these four DD bands were murine Ring3, sema 4g, GCS, and p45 NF E2. The DD results suggested that their expression may have been up-regulated during P. yoelii infection. PCR was then performed using gene-specific primers shown in Table I on freshly synthesized UL and IL cDNA. The amplification results demonstrated that these transcripts were constitutively expressed in UL, although quantitative expression levels were not determined (data not shown).

To measure changes in gene expression of these transcripts via a semiquantitative RT-PCR, cycle titration RT-PCR was first performed to determine the exponential portion of the amplification curve for each transcript (9). Fig. 1 shows the plotted exponential curves of β-actin, sema 4g, and TNF-α in the UL cDNA. The PCR cycle number that corresponded to half the maximal amplification was determined for each gene product from the liver, spleen, and kidney tissue samples and is shown in Table II. cDNA from UL and IL was subsequently amplified for the appropriate number of cycles using gene-specific primers for Ring3, sema 4g, GCS, p45 NF E2, IFN-γ, and TNF-α. Transcript values (measured as counts of [32P]deoxyribocytidine incorporation) were then normalized to those of β-actin for each tissue tested. Because IFN-γ has been shown to participate in the inhibition of liver stage parasites in immunized animals (10), and TNF-α expression has been shown to be inversely correlated with that of GCS (11), transcript levels of these two cytokines were also measured.

There was a 2-fold increase in expression levels of sema 4g and p45 NF E2 compared with that in UL cDNA, whereas a 3- and 4-fold increase in GCS and Ring3 expression levels were detected in the IL cDNA. On the contrary, IFN-γ and TNF-α levels were both down-regulated in IL cDNA. IFN-γ expression was decreased by 68%, whereas that of TNF-α was decreased by 64% relative to that of the UL levels (Fig. 2).

FIGURE 2.

Comparison of murine transcript levels between UL and IL. Dotted line represents the sham values, which are set at 100%. Values are mean ± SEM where n = 6 separate infections. ∗, Statistical significance based on Student’s t test with p ≤ 0.05.

FIGURE 2.

Comparison of murine transcript levels between UL and IL. Dotted line represents the sham values, which are set at 100%. Values are mean ± SEM where n = 6 separate infections. ∗, Statistical significance based on Student’s t test with p ≤ 0.05.

Close modal

To understand whether the host transcripts were responding to the parasite infection in an organ-specific manner, spleens and kidneys from 24 h P. yoelii-infected animals were collected and processed. The Ring3 expression level in spleens of infected mice increased by ∼30%, whereas sema 4g, GCS, and p45 NF E2 were not detected. Splenic IFN-γ expression from parasite-infected mice, was 10 times higher than in the UL sample. However, TNF-α expression level decreased by 30% (Fig. 3). In the kidney, Ring3 and sema 4g expression was 6 and 4 times greater than that in the UL, respectively, whereas p45 NF E2 and IFN-γ were not detected. Lastly, TNF-α was down-regulated by 44% in the kidney (Fig. 4).

FIGURE 3.

Comparison of murine transcript levels between sham-infected and 24 h Plasmodium yoelii-infected spleens. Dotted line represents the sham values, which are set at 100%. Values are mean ± SEM where n = 6 separate infections. ∗, Statistical significance based on Student’s t test with p ≤ 0.05.

FIGURE 3.

Comparison of murine transcript levels between sham-infected and 24 h Plasmodium yoelii-infected spleens. Dotted line represents the sham values, which are set at 100%. Values are mean ± SEM where n = 6 separate infections. ∗, Statistical significance based on Student’s t test with p ≤ 0.05.

Close modal
FIGURE 4.

Comparison of murine transcript levels between sham-infected and 24 h Plasmodium yoelii-infected kidneys. Dotted line represents the sham values, which are set at 100%. Values are mean ± SEM where n = 6 separate infections. ∗, Statistical significance based on Student’s t test with p ≤ 0.05.

FIGURE 4.

Comparison of murine transcript levels between sham-infected and 24 h Plasmodium yoelii-infected kidneys. Dotted line represents the sham values, which are set at 100%. Values are mean ± SEM where n = 6 separate infections. ∗, Statistical significance based on Student’s t test with p ≤ 0.05.

Close modal

To determine whether these observations were unique responses to Plasmodium infection or generic responses to hepatic intracellular invasion, livers from mice infected with a related Apicomplexan parasite, T. gondii, were analyzed for changes in the same transcript levels. Although there was no difference in GCS, sema 4g, and p45 NF E2 expressions between UL and IL levels, Ring3 levels in IL were 4 times greater than that in the UL samples. This increase was similar to that seen in the Plasmodium-infected livers. In contrast to P. yoelii-infected livers, sema 4g transcript levels decreased by 68% in the T. gondii-infected livers. IFN-γ and TNF-α levels increased by ∼6- and 40-fold, respectively, to those of the controls (Fig. 5).

FIGURE 5.

Comparison of murine transcript levels between sham-infected and 24 h T. gondii-infected livers. Values are mean ± SEM where n = 2. Statistical analysis could not be performed on this set of data.

FIGURE 5.

Comparison of murine transcript levels between sham-infected and 24 h T. gondii-infected livers. Values are mean ± SEM where n = 2. Statistical analysis could not be performed on this set of data.

Close modal

The liver is responsible for multiple functions including the detoxification of compounds (12). It is equipped with mechanisms to protect the host from infectious diseases, toxins, and other unwarranted and harmful products. Therefore, it is reasonable to expect that the expression of particular genes under stressful conditions such as elevated temperature, chemical intoxication, and infection would be altered. For example, host macrophages in the liver express heat shock protein 65 to prevent apoptosis of Toxoplasma-infected cells (13). Likewise, there are a number of reports demonstrating the presence of host responses to Plasmodium liver infection. Nussler et al. showed that the liver responded to Plasmodium infection by increasing the production of C-reactive protein induced by IL-1 stimulation (14). Recently, when Pied et al. demonstrated that a subpopulation of γδT cells, CD4CD8NK1.1+TCR-αint, were up-regulated during the acute blood stage infection, they also reported that these NK1.1+ cells were able to inhibit parasite growth inside hepatocytes, and their killing effect was reversed by anti-CD3 Ab (15). Evidence strongly suggests that specific host responses occur during liver stage infection and may represent the attempt by the host to control the infection. This study further suggests that there are at least several host transcripts whose expression pattern is altered due to Plasmodium liver infection.

Ring3 is localized to the MHC class II region in all species studied and, thus, it may participate in MHC presentation or gene expression (16). Ring3 is constitutively expressed in the liver, testis, lung, heart, and kidney (17). Limited functional analysis has revealed that Ring3 may be a serine/threonine kinase localized exclusively in the nucleus; however, a recent article by Platt et al. suggests that Ring3 is not a kinase but appears to recruit an as yet unidentified serine/threonine protein kinase into its complex (18). Expression of Ring3 is elevated upon cell proliferation and in patients with acute and chronic lymphocytic leukemia (19). Currently, the genomic sequence of the mouse Ring3 has been completed but the exact physiological function of Ring3 and its potential of being a kinase remain unknown. Although the significance of the increase of Ring3 expression in livers, spleens, and kidneys in P. yoelii-infected mice is equally unclear, its association with MHC class II suggests that it may be involved in the immune response of the host. The up-regulation of Ring3 expression in both Plasmodium-infected liver and kidney was much more dramatic than that in the infected spleen. Further investigation into the basic function of Ring3 is required to explain this phenomenon. One possibility could be that the liver and the kidney were simply more responsive than the spleen to changes resulting from the Plasmodium infection. In addition, the up-regulation of Ring3 was also observed in T. gondii-infected liver, suggesting that the increased level of Ring3 mRNA may be a generic and systemic host response to parasitic infection.

Sema 4g expression was similarly elevated in livers and kidneys but not expressed in spleens of P. yoelii-infected mice. This was in agreement with the recently published work by Li et al., which also found sema 4g to be expressed only in liver, kidney, and brain (19). Interestingly, infection with T. gondii did not produce a change in the expression of sema 4g mRNA. Thus, the increase of sema 4g expression appeared to be parasite specific. However, the up-regulation of sema 4g expression in the infected livers was not statistically significant; therefore, we concluded that the up-regulation of sema 4g expression level appeared to be kidney specific. The semaphorin family contains secreted and transmembrane signal proteins that function in the nervous, cardiovascular, and immune systems. Sema 4g was first isolated in the mouse (20) where the protein contains semaphorin, a single putative Ig-like transmembrane, and cytoplasmic domains. Sema 4g belongs to semaphorin subclass 4, which is expressed in high levels in lymphoid tissues, unlike other members of the semaphorin family (21). Therefore, immune cells expressing sema 4g may be involved in the P. yoelii liver infection. McKenna et al. (4) recently reported that γδT cells participate in the early response to the exoerythrocytic stage of the malarial infection. It is reasonable to suggest that other immune cells may be involved as well. Thus, the increased sema 4g expression could reflect the expression of the gene in activated immune cells within the kidney vasculature.

GCS initiates the synthesis of glutathione, a major cellular antioxidant. Glutathione is involved in regulating the redox status of cells (22). GCS is a heterodimer made up of a catalytic (H chain) and a regulatory (L chain) subunit. Changes in GCS activity can result from regulation at either the transcriptional or posttranslational level, affecting only the heavy subunit or both of the subunits. Conditions such as drug resistance, hormonal influences, oxidative stress, and treatment with antioxidants have all been shown to affect GCS expression and activity (22). Manna et al. (11) reported that the overexpression of GCS blocked the effect of TNF-α on NF-κB activation, cytoplasmic I-κB degradation, nuclear translocation of p65, NF-κB-dependent gene transcription, and, most importantly, TNF-α-mediated cytotoxicity and caspase 3 activation. The simultaneous up-regulation in GCS expression and down-regulation of IFN-γ and TNF-α in P. yoelii-infected livers are consistent with the successful development of mature parasites during this stage of malarial infection. Type 1 cytokine responses, which include these two cytokines, play a crucial role in the clearing of the exoerythrocytic stage of the malaria parasites (10, 23). The diminished expression levels of both cytokines could potentially promote parasite survival in the liver. NF-κB-dependent gene transcription includes ICAM-1, VCAM-1, and E-selectin. The down-regulation of NF-κB-dependent gene transcription due to the overexpression of GCS (11) may potentially contribute to the lack of neutrophil aggregation in the P. yoelii-infected liver (24) because these adhesion molecules are involved in the recruitment of neutrophils to sites of infection.

As expected, both IFN-γ and TNF-α transcript levels were substantially elevated in Toxoplasma-infected livers. Experimentally, one of the hallmark characteristics of Toxoplasmosis is the elevated levels of IFN-γ and TNF-α, which prevent uncontrolled parasite growth and, consequently, host mortality (25). These cytokines induce the production of NO, which is responsible for the elimination of the parasites. Toxoplasma-infected livers frequently appear discolored and display occasional foci of inflammatory cells that are mediated by IFN-γ and TNF-α. The pathologic responses generated in Toxoplasma-infected livers do not occur in Plasmodium-infected livers, so it might be expected that IFN-γ and TNF-α expression would not be up-regulated in a Plasmodium-infected mouse.

IFN-γ expression levels in the spleen were elevated, whereas the TNF-α levels were reduced. This was unexpected because these cytokines are usually coordinately expressed. This discordance cannot currently be explained, but may be due to the differential responses of tissues to P. yoelii infection. Alternatively, because a large dose of P. yoelii sporozoites were inoculated into each animal, the possibility exists that some sporozoites may be cleared by the spleen, thus generating an immune response independent to the liver.

p45 NF E2 is a member of the basic leucine zipper family of dimeric transcription factors. It consists of a widely expressed 18-kDa subunit and a tissue-restricted 45-kDa subunit (26). p45 NF E2 is known to function in the regulation of globin gene transcription and platelet production. Its expression level is reduced in apoptotic cells, although its binding capability to DNA is not altered (27). The up-regulation of p45 NF E2 in Plasmodium-infected livers but not in T. gondii livers is suggestive of an underlying anti-apoptotic and adaptive mechanism of infection inherent to P. yoelii.

Clearly, host responses generated as a result of Plasmodium infection exist (28, 29, 30, 31, 32). For instance, GPI is a potent Plasmodium glycolipid toxin that not only induces IL-1 production by macrophages during the blood stage of the infection, but also regulates glucose metabolism in adipocytes, resulting in profound hypoglycemia (28). It is postulated that GPI may be responsible for pleiotropic effects on a variety of host cells by substituting for the endogenous GPI second messenger signaling pathway of the host (29). Therefore, it is possible that the differential regulation of the reported host genes in P. yoelii-infected livers may be caused by parasite-derived material that affects the host locally or systemically. Further studies are needed to determine how Plasmodium actively influences intracellular processes that ultimately control gene transcription within infected cells of other tissues. Nevertheless, the changes in expression levels of many host gene products may facilitate parasite development and promote the survival of the parasite.

We thank Dr. B. D. Rodgers for technical assistance and Dr. George Yap for providing T. gondii-infected tissues.

1

This work was supported by grants from the National Institutes of Health (T32AI075) and Naval Medical Research and Development Command (work units STOF6.161102AA0101BFX, STO F6.262787A00101EFX, and STEPC611102A0101BCX).

3

Differentially displayed bands were detected exclusively in the IL samples.

4

Abbreviations used in this paper: Ring3, Really Interesting New Gene 3; GCS, glutamylcysteine synthetase; p45 NF E2, p45 NF erythroid 2; sema 4g, semaphorin subclass 4 member G; DD, differential(ly) display(ed); UL, sham-infected livers; IL, 24 h P. yoelii-infected livers.

5

Abbreviations used in this paper: Ring3, really interesting new gene 3; GCS, glutamylcysteine synthetase; p45 NF E2, p45 NF erythroid 2; sema 4g, semaphorin subclass 4 member G; DD, differential(ly) display(ed); UL, sham-infected livers; IL, 24 h P. yoelii-infected livers.

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