Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum.

A major obstacle in the global effort to control malaria is the paucity of anti-malarial drugs. This is compounded by the continuing emergence and spread of resistance to old and new anti-malarial drugs in the malarial parasites. Here we describe the anti-malarial effect of phosphorothioate antisense (AS) oligodeoxynucleotides (ODNs) targeting the aldolase enzyme of Plasmodium falciparum, using the asexual blood stages of the parasite grown in vitro. The blood stages of P. falciparum depend almost entirely on the energy produced by their own glycolysis. Aldolase, the fourth enzyme of the glycolytic pathway, is highly upregulated during the malarial 48-h life cycle. We found that the mRNA of this enzyme can be inhibited, in a sequence specific manner, using AS-ODN to the splice sites on the pre-mRNA of malarial aldolase. At the enzyme level, both specific AS-ODNs for the splice sites, as well as for the translation initiation site on mature mRNA, can inhibit aldolase enzyme activity within the trophozoites of P. falciparum. Furthermore, this downregulation of the malarial aldolase results in a reduction in the production of ATP within the parasite. Finally, the treatment reduces parasitemia. In summary, AS-ODNs targeting the aldolase gene of P. falciparum can interfere with the blood-stage life cycle of this parasite in vitro by inhibiting the expression of the enzyme aldolase which results in decreased malarial glycolysis and energy production. Thus, we conclude that blockade of the expression of malarial glycolytic enzymes using specific AS-ODNs has the potential of a new anti-malarial strategy.

Transgenic mice expressing human fetal globin are protected from malaria by a novel mechanism.

Studies in vitro by Pasvol et al (Nature, 270:171, 1977) have indicated that the growth of Plasmodium falciparum in cells containing fetal hemoglobin (HbF = alpha2gamma2) is retarded, but invasion is increased, at least in newborn cells. Normal neonates switch from about 80% HbF at birth to a few percent at the end of the first year of life. Carriers of beta-thalassemia trait exhibit a delay in the normal HbF switch-off, which might partially explain the protection observed in populations with this gene. To study this hypothesis in vivo, we used transgenic (gamma) mice expressing human Agamma and Ggamma chains resulting in 40% to 60% alpha2Mgamma2 hemoglobin, infected with rodent malaria. Two species of rodent malaria were studied. P chabaudi adami causes a nonlethal infection, mainly in mature red blood cells (RBC). P yoelii 17XNL is a nonlethal infection, invading primarily reticulocytes, whereas P yoelii 17XL is a lethal variant of P yoelii 17XNL and causes death of mice in approximately 1 to 2 weeks. Data indicate that this strain may cause a syndrome resembling cerebral malaria caused by P falciparum (Am J Trop Med Hyg, 50:512, 1994). In gamma transgenic mice infected with P chabaudi adami, the parasitemia rose more quickly (in agreement with Pasvol) than in control mice, but was cleared more rapidly. In mice infected with P yoelii 17XNL, a clear reduction in parasitemia was observed. Interestingly, splenectomy before this infection, did not reverse protection. The most striking effect was in lethal P yoelii 17XL infection. Control mice died between 11 to 13 days, whereas gamma mice cleared the infection by day 22 and survived, a phenomenon also observed in splenectomized animals. These results suggest that HbF does indeed have a protective effect in vivo, which is not mediated by the spleen. In terms of mechanisms, light microscopy showed that intraerythrocytic parasites develop slowly in HbF erythrocytes, and electron microscopy showed that hemozoin formation was defective in transgenic mice. Finally, digestion studies of HbF by recombinant plasmepsin II demonstrated that HbF is digested only half as well as hemoglobin A (HbA). We conclude that HbF provides protection from P falciparum malaria by the retardation of parasite growth. The mechanism involves resistance to digestion by malarial hemoglobinases based on the data presented and with the well-known properties of HbF as a super stable tetramer. In addition, the resistance of normal neonates for malaria can now be explained by a double mechanism: increased malaria invasion rates, reported in neonatal RBC, will direct parasites to fetal cells, as well as F cells, and less to the approximately 20% of HbA containing RBC, amplifying the antimalarial effects of HbF.

Microvascular hemodynamics and in vivo evidence for the role of intercellular adhesion molecule-1 in the sequestration of infected red blood cells in a mouse model of lethal malaria.

The cytoadherence of infected red blood cells (IRBCs) to the vascular endothelium is the major cause of IRBC sequestration and vessel blockage in the cerebral form of human malaria. Among the rodent models of malaria, Plasmodium yoelii 17XL-infected mice show many similarities with the human cerebral malaria caused by P. falciparum. In both, the sequestration of IRBCs in the brain vessels is secondary to the cytoadherence of IRBCs to the vascular endothelium. Similar to P. falciparum infection in the human but in contrast to P. berghei ANKA infection in mice, P. yoelii 17XL results in little, if any, accumulation of monocytes in the brain. In vivo microcirculatory studies reported here were designed to further understand the hemodynamic aspects and mechanisms underlying cytoadherence of IRBCs in the P. yoelii model using the easily accessible cremaster muscle vasculature. The results show significant decreases in arteriovenous red blood cell velocities (Vrbc) and wall shear rates in the microcirculation of P. yoelii-infected mice, with a maximal decrease occurring in small-diameter postcapillary venules, the main sites of cytoadherence. This reflects contributions from IRBC cytoadherence as well as from increased rigidity of parasitized red blood cells. No cytoadherence is observed in arterioles of the infected mice despite decreased wall shear rates, indicating that endothelial receptors for cytoadherence are restricted to venules. Infusion of a monoclonal antibody (MAb) against the intercellular adhesion molecule-1 (ICAM-1) resulted in significant increases in both arteriolar and venular Vrbc and wall shear rates, accompanied by detachment of adhered IRBCs at some venular sites. The peripheral blood smears taken after the MAb infusion showed a distinct increase in the percentage of schizonts, again indicating detachment and/or prevention of cytoadherence. An MAb against the vascular cell adhesion molecule-1 (VCAM-1) as well as an irrelevant control antibody had no effect on these parameters. These results provide the first in vivo microcirculatory evidence indicating involvement of ICAM-1, but not of VCAM-1, in the sequestration of IRBCs in a rodent model of cerebral malaria.

Correlation of increased expression of intercellular adhesion molecule-1, but not high levels of tumor necrosis factor-alpha, with lethality of Plasmodium yoelii 17XL, a rodent model of cerebral malaria.

Previous studies demonstrated that Plasmodium yoelii 17XL, a lethal strain of rodent malaria, causes a syndrome in SW mice that resembles human cerebral malaria. The mouse brain pathology is characterized by cytoadherence of parasitized erythrocytes. Here, the possible mechanisms mediating cerebral malaria in this model were studied and the results were compared with a nonlethal strain of this parasite, P. yoelii 17XNL (nonlethal), which does not cause cerebral malaria. Immunostaining for intercellular adhesion molecule-1 (ICAM-1) revealed an increase in expression of this protein in the small venules and capillaries of the brains of infected mice that increased with time after infection. Staining was more pronounced during the lethal infection than the nonlethal infection. Some staining with monoclonal antibody to vascular cell adhesion molecule-1 was also observed, but it was quantitatively less than ICAM-1 staining and was limited to larger venules. During the lethal infection, levels of tumor necrosis factor-alpha (TNF-alpha) increased rapidly, peaking on day 4. In contrast, mice infected with nonlethal P. yoelii had a slower serum TNF-alpha response that peaked on day 10, prior to the maximum parasitemia. In addition, mice with a targeted disruption of the TNF-alpha gene (TNF-alpha-/- mice) were infected with the lethal and nonlethal strains of P. yoelii 17X. The TNF-alpha-/- mice infected with the nonlethal parasite had significantly higher levels of parasitemia than controls, whereas TNF-alpha-/- mice infected with the lethal strain had slightly higher levels of infected erythrocytes but were equally susceptible to death from this infection. Thus, TNF-alpha does not appear to be essential in mediating death. These results demonstrate that P. yoelii 17XL infection has features in common with human cerebral malaria and suggest that this model may be useful in testing strategies to alleviate this syndrome.

A first evaluation of the natural high molecular weight polymeric Lumbricus terrestris hemoglobin as an oxygen carrier.

Lumbricus terrestris hemoglobin (LtHb), an unusually stable Hb (MW approximately 4x10(6) Da) with respect to dissociation and oxidation, circulates extracellularly in the earthworm and at neutral pH exhibits oxygen affinity and cooperativity similar to that of human HbA. Results suggest that LtHb may serve as a model for a high molecular weight extracellular oxygen carrier. Mice and a rat model partially exchanged with LtHb showed no apparent behavioral and physical changes. 31P NMR spectroscopy of perfused guinea pig hearts, used to assess phosphocreatine levels as an indication of the ability of LtHb to serve as an oxygen carrier to the heart, demonstrated that LtHb provides oxygen to the tissue and maintains the energy metabolism significantly better than the control non-Hb perfusion media. One day after infusion, video enhanced microscopy imaging of the mice cremaster muscle vasculature reveals temporal adhesion of leukocytes to the endothelial walls with temporal infiltration of leukocytes to the surrounding tissue, correlated with dosage. Exchanged mice rechallenged with LtHb show no overt allergic response or death. Further evaluation of this natural extracellular Hb as a potential polymeric Hb blood substitute/perfusion agent is warranted.

Protection from lethal malaria in transgenic mice expressing sickle hemoglobin.

Previous studies from our laboratories have shown that transgenic mice expressing high levels of beta S globin are well-protected from Plasmodium chabaudi adami and partially protected against P berghei (Shear et al, Blood 81:222, 1993). We have now infected transgenic mice expressing low (39%), intermediate (57%), and high (75%) levels of beta S with the virulent strain of P yoelii (17XL) that appears to cause cerebral malaria. We find that the level of protection in these three groups of mice correlates positively with the level of beta S chain expression in the mice. Seven of nine mice expressing the high level of beta S recovered from infection, as did 7 of 9 mice expressing the intermediate level of beta S. Control mice and mice expressing the lower level of beta S all succumbed to infection. In mice expressing high and intermediate levels of beta S, parasites were found almost exclusively in reticulocytes during recovery, suggesting that mature red blood cells expressing beta S are more resistant than reticulocytes. These studies confirm epidemiologic data and offer insight into the mechanism of protection of sickle trait individuals against falciparum malaria.

Cerebral malaria in mice: demonstration of cytoadherence of infected red blood cells and microrheologic correlates.

To understand the microcirculatory events during cerebral malaria, we have studied the lethal strain of rodent Plasmodia, Plasmodium yoelii 17XL, originally described by Yoeli and Hargreaves in 1974. The virulence of P. yoelii 17XL is caused by intravascular sequestration of infected red blood cells (IRBCs), especially in the brain vessels and capillaries. This mouse model resembles human P. falciparum infection more closely than P. berghei ANKA infection since it shows little, if any, inflammation of the brain. In vivo microcirculatory studies on cytoadherence of IRBCs were performed using the cremaster muscle preparation, which is an easily accessible vasculature for intravital observations. Ex vivo assay of cytoadherence was carried out in the artificially perfused mesocecum preparation of the rat. The results in either preparation demonstrated cytoadherence of IRBCs that was restricted to postcapillary venules. Furthermore, the in vivo measurements showed the prevalence of cytoadherence in small-diameter (< 40 microns) venules in accordance with the local wall shear rates. The parasitized animals demonstrated significantly reduced red blood cell velocities and wall shear rates in the small-diameter postcapillary venules of the cremaster. The relationship between cytoadherence and venular wall shear rates was also reflected in the inverse correlation between the number of adhered cells and the venular diameter in the ex vivo mesocecum preparation. In the ex vivo preparation, cytoadherence of IRBCs was accompanied by a higher peripheral resistance. Transmission electron microscopy of the cremaster muscle and brain tissues showed a tight association of IRBCs with the endothelium of small venules. These observations demonstrate that cytoadherence of P. yoelii 17XL-infected mouse red blood cells is very similar to that of P. falciparum-infected cells. Thus, this model should allow a detailed analysis of the molecular mechanisms involved in the generation of cerebral malaria by cytoadherence of the infected red blood cells to the vascular endothelium.

Transgenic mice expressing human sickle hemoglobin are partially resistant to rodent malaria.

The polymorphic frequency of the gene for beta s-globin involved in the generation of sickle trait and sickle cell anemia in the human population is caused by the enhanced resistance of sickle trait individuals to Plasmodium falciparum malaria, as supported by epidemiologic and in vitro studies. However, the mechanism for the protective effect of sickle hemoglobin in vivo has not been fully defined. The generation of transgenic mice expressing high levels of human beta s- and alpha-chains has allowed us to study this phenomenon in vivo in an experimental model. We infected the transgenic beta s mice with two species of rodent malaria and found a diminished and delayed increase in parasitemia as compared with controls. This is in contrast to our previous studies involving the introduction of a beta A transgene, which does not alter the infection. The use of this model allowed us to address the question of the mechanism of protection against malaria in mice expressing sickle hemoglobin. We find that splenectomy of transgenic mice completely reverses the protection against Plasmodium chabaudi adami infection. The results reported have shown a relationship between the presence of the beta s gene product and partial resistance to malaria in an experimental model in vivo and shows that the spleen plays an important role in this protection.

Transgenic and mutant animal models to study mechanisms of protection of red cell genetic defects against malaria.

Malaria, caused by members of the genus Plasmodia, is still the most prevalent parasitic disease in the world. In an attempt to understand genetic factors conferring resistance to malaria, mouse models of thalassemia, sickle trait, and ankyrin and spectrin deficiency were studied during infection with species of malaria infectious to rodents. Although growth of P. falciparum is not inhibited in thalassemic erythrocytes in culture, mice carrying a beta-thalassemia mutation were protected from Plasmodium chabaudi adami, supporting epidemiologic findings. Transgenic mice expressing beta s hemoglobin were also significantly protected from two species of rodent malaria. Importantly, a significant role for the spleen in protection in the beta s transgenic mice was found. Finally, mice deficient in spectrin and ankyrin were studied with respect to their ability to support the growth of malaria. It was found that spectrin deficient mice were almost completely refractory to P. chabaudi adami and P. berghei. These models will allow further study of host factors in resistance to malaria.

Resistance to malaria in ankyrin and spectrin deficient mice.

Inbred mice carrying mutations in ankyrin and/or spectrin synthesis and assembly were studied for their ability to support the growth of the rodent malarias, Plasmodium chabaudi adami and P. berghei, in vivo. Mice carrying the nb/nb (normoblastosis) mutation which do not synthesize ankyrin and therefore also have a deficiency in membrane-bound spectrin, were refractory to P. chabaudi adami, which invades mature erythrocytes and to P. berghei, which invades reticulocytes. Similarly, sph/sph mice which do not synthesize the alpha chain of spectrin but do synthesize ankyrin, were also resistant to both parasites. The heterozygote for the nb defect (nb/+) exhibited a diminution of parasitaemia. We conclude that the host cell spectrin may be necessary for the invasion and/or growth of rodent malarial parasites.

Cytokine production in lethal and non-lethal murine malaria.

Levels of IFN-gamma, IL-2 and IL-4 were measured in vitro during the course of non-lethal Plasmodium chabaudi adami and lethal P. chabaudi strain 1309 infections in BALB/cByJ mice. Spleen cells from mice infected with the non-lethal Plasmodium had a higher initial response to P. chabaudi antigens than mice infected with P. chabaudi strain 1309, as determined by measuring all three lymphokines. We conclude that both Th1 and Th2 subsets of T helper lymphocytes are activated during P. chabaudi adami infection but that these responses are suppressed in mice infected with the more virulent P. chabaudi strain 1309.

Enhanced treatment of Pneumocystis carinii pneumonia in rats with interferon-gamma and reduced doses of trimethoprim/sulfamethoxazole.

Interferon-gamma (IFN-gamma) was used to treat rats with steroid-induced Pneumocystis carinii pneumonia (PCP). Treatment with 427,000 U/day prophylactically prevented infection in this model. Treatment with 200,000 U, three times/week for 2 weeks caused a significant reduction in the number of cysts in the lungs and prolonged survival of the rats. In addition, IFN-gamma and trimethoprim/sulfamethoxazole behaved synergistically in the treatment of PCP in rats. Reduced dosages of each drug, when given together, caused an almost complete eradication of the infection. This may be a useful approach in patients with acquired immune deficiency syndrome in whom anti-Pneumocystis drugs are often toxic.

Role of IFN-gamma in lethal and nonlethal malaria in susceptible and resistant murine hosts.

IFN-gamma plays an important role in host defense against microbial disease. Here, we studied the role of IFN-gamma in lethal and nonlethal murine malaria. Administration of recombinant murine IFN-gamma resulted in a dose-dependent protection of SW, BALB/cByJ, and CBA/J mice from the lethal variant of Plasmodium yoelii 17x (PyL) but had little effect on the course of the nonlethal variant of this parasite (PyNL). Administration of recombinant IFN-gamma also resulted in the activation of peritoneal macrophages for increased phagocytosis of malaria-infected erythrocytes and release of H2O2, as measured in vitro. The ability of spleen cells from infected mice to produce endogenous IFN-gamma and release H2O2 during the course of malaria was also studied. In BALB/cByJ mice, which are relatively susceptible to PyL and PyNL, there was an initial burst of IFN-gamma only in response to PyNL whereas in CBA/J mice, which are relatively resistant to these parasites, there was an initial burst of IFN-gamma in response to both PyL and PyNL. The kinetics of H2O2 release corresponded to that of IFN-gamma. In all infections, levels of IFN-gamma declined as parasitemia increased; however, nonlethal infections were characterized by a recovery of both IFN-gamma activity and H2O2 release as parasitemia declined. These data suggest that IFN-gamma may play an important role in modulating the course of malaria infections by activating macrophages for both intracellular and extracellular parasite destruction.

Variation in expression of antibody-dependent cell-mediated cytotoxicity in rodents with malaria.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against mouse erythrocytes sensitized with immunoglobulin G was studied in mice with malaria. Spleen cells from mice had enhanced cytotoxic activity early in Plasmodium berghei infection but not later in the disease. Sera from infected animals and partially purified malarial immune complexes inhibited ADCC. In addition, ADCC was diminished in spleen cells from mice infected with the lethal variant of P. yoelii 17x compared with that in mice infected with the nonlethal variant. P. berghei-infected erythrocytes did not release 51Cr when incubated with effector cells unless the erythrocytes were sensitized with antibodies against normal mouse erythrocytes.

Malaria in beta-thalassemic mice and the effects of the transgenic human beta-globin gene and splenectomy.

To investigate the protective effects of beta-thalassemia against malaria, rodent malaria parasites were studied in C57BL/6J mice with beta-thalassemia, in mice in which the thalassemia had been transgenically corrected with the human beta A-globin gene, and in hematologically normal mice. In thalassemic mice, Plasmodium chabaudi adami infection was inhibited and peak parasitemia was variably delayed. In transgenically corrected mice, infection proceeded as in normal mice. Plasmodium berghei infection proceeded more rapidly in thalassemic mice, but survival was not different. Splenectomized normal mice displayed high-level parasitemia that peaked twice and persisted as a low-level parasitemia for more than 20 days after normal intact mice were free of all parasites. Splenectomized thalassemic mice showed a delay of 5 days in attaining peak parasitemia, but the parasitemia persisted as in normal splenectomized mice. Thus, for P. chabaudi, which displayed no preference for immature erythrocytes, beta-thalassemia offers enhanced resistance for the host. However, for P. berghei, which preferentially invades reticulocytes, thalassemia is not protective. The protective effects of the normal mouse spleen were observed, but the paradoxical facilitation of parasite growth by the thalassemic spleen is a new finding that will require further experimentation to explain. This new in vivo laboratory documentation of thalassemic protection against some rodent malaria parasites may serve as a useful model in further efforts to control this major infectious disease.

Protective antigen in the membranes of mouse erythrocytes infected with Plasmodium chabaudi.

A malarial antigen, Pc96, in the plasma membrane of erythrocytes infected with Plasmodium chabaudi has been identified. It is synthesized by the parasite and present during most of the growth stages of the intra-erythrocytic cycle as demonstrated by immunofluorescence. The antigen has a molecular weight of approximately 96,000. Monoclonal antibodies raised against this antigen were used to isolate the protein by affinity chromatography. Mice immunized with affinity-purified Pc96 were partially protected against blood induced-P. chabaudi infection. This result indicates the existence of a protective antigen in the membranes of erythrocytes parasitized by a rodent malaria and encourages the search for analogous antigens in human malaria parasites as possible candidate molecules for malaria vaccination.

Human and primate malarial sera inhibit Fc receptor-mediated phagocytosis.

Sera obtained from humans in P. falciparum-endemic regions and from P. vivax-infected Saimiri sciureus were assayed for their ability to inhibit Fc receptor-mediated phagocytosis. Some sera of humans exposed to P. falciparum from The Gambia, Sudan, and Thailand inhibited ingestion via the Fc receptor by normal human monocytes. In addition, sera from infected monkeys and a high molecular weight fraction of infected monkey serum inhibited ingestion of EIgG by normal monkey spleen macrophages. Generally, inhibition was correlated with higher parasitemia and higher IFA titers.