α-Galactosylceramide-activated Vα14 natural killer T cells mediate protection against murine malaria

Natural killer T (NKT) cells are a unique population of lymphocytes that coexpress a semiinvariant T cell and natural killer cell receptors, which are particularly abundant in the liver. To investigate the possible effect of these cells on the development of the liver stages of malaria parasites, a glycolipid, α-galactosylceramide (α-GalCer), known to selectively activate Vα14 NKT cells in the context of CD1d molecules, was administered to sporozoite-inoculated mice. The administration of α-GalCer resulted in rapid, strong antimalaria activity, inhibiting the development of the intrahepatocytic stages of the rodent malaria parasites Plasmodium yoelii and Plasmodium berghei. The antimalaria activity mediated by α-GalCer is stage-specific, since the course of blood-stage-induced infection was not inhibited by administration of this glycolipid. Furthermore, it was determined that IFN-γ is essential for the antimalaria activity mediated by the glycolipid. Taken together, our results provide the clear evidence that NKT cells can mediate protection against an intracellular microbial infection.

Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites.

Immunization of rodents and humans with irradiation-attenuated malaria sporozoites confers preerythrocytic stage-specific protective immunity to challenge infection. This immunity is directed against intrahepatic parasites and involves T cells and interferon gamma, which prevent development of exoerythrocytic stages and subsequent blood infection. The present study was undertaken to determine how protective immunity is achieved after immunization of rodent hosts with irradiated Plasmodium berghei sporozoites. We present evidence that irradiated parasites persist in hepatocytes of rats and mice for up to 6 months after immunization. A relationship between the persistence of parasites and the maintenance of protective immunity was observed. Protective immunity was abrogated in irradiated-sporozoite-immunized rats following the application of chemotherapy to remove preexisting liver parasites. Additionally, protective immunity against sporozoite challenge was established in rats vaccinated with early and late hepatic stages of irradiated parasites. These results show that irradiation-attenuated sporozoites produce persistent intrahepatic stages in vivo necessary for the induction and maintenance of protective immunity.

Estimating sequestered parasite population dynamics in cerebral malaria

Clinical investigation of malaria is hampered by the lack of a method for estimating the number of parasites that are sequestered in the tissues, for it is these parasites that are thought to be crucial to the pathogenesis of life-threatening complications such as cerebral malaria. We present a method of estimating this hidden population by using clinical observations of peripheral parasitemia combined with an age-structured mathematical model of the parasite erythrocyte cycle. Applying the model to data from 217 Gambian children undergoing treatment for cerebral malaria we conclude that although artemether clears parasitemia more rapidly than quinine, the clearance of sequestered parasites is similar for the two drugs. The estimated sequestered mass was found to be a more direct predictor of fatal outcome than clinically observed parasitemia. This method allows a sequential analysis of sequestered parasite population dynamics in children suffering from cerebral malaria, and the results offer a possible explanation for why artemether provides less advantage than might have been expected over quinine in reducing mortality despite its rapid effect on circulating parasites.

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-γ-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-γ production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-γ production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [3H]-5-HT-moduline bound specifically to rat brain sections with high affinity (Kd = 0.77 nM and Bmax = 0.26 dpm/mm2). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT1B receptor knock-out mice, the specific binding of [3H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.

Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites

Immune responses of the malaria vector mosquito Anopheles gambiae were monitored systematically by the induced expression of five RNA markers after infection challenge. One newly isolated marker encodes a homologue of the moth Gram-negative bacteria-binding protein (GNBP), and another corresponds to a serine protease-like molecule. Additional previously described markers that respond to immune challenge encode the antimicrobial peptide defensin, a putative galactose lectin, and a putative serine protease. Specificity of the immune responses was indicated by differing temporal patterns of induction of specific markers in bacteria-challenged larvae and adults, and by variations in the effectiveness of different microorganisms and their components for marker induction in an immune-responsive cell line. The markers exhibit spatially distinct patterns of expression in the adult female mosquito. Two of them are highly expressed in different regions of the midgut, one in the anterior and the other in the posterior midgut. Marker induction indicates a significant role of the midgut in insect innate immunity. Immune responses to the penetration of the midgut epithelium by a malaria parasite occur both within the midgut itself and elsewhere in the body, suggesting an immune-related signaling process.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

Different response to Plasmodium falciparum malaria in West African sympatric ethnic groups

The comparison of malaria indicators among populations that have different genetic backgrounds and are uniformly exposed to the same parasite strains is one approach to the study of human heterogeneities in the response to the infection. We report the results of comparative surveys on three sympatric West African ethnic groups, Fulani, Mossi, and Rimaibé, living in the same conditions of hyperendemic transmission in a Sudan savanna area northeast of Ouagadougou, Burkina Faso. The Mossi and Rimaibé are Sudanese negroid populations with a long tradition of sedentary farming, while the Fulani are nomadic pastoralists, partly settled and characterized by non-negroid features of possible caucasoid origin. Parasitological, clinical, and immunological investigations showed consistent interethnic differences in Plasmodium falciparum infection rates, malaria morbidity, and prevalence and levels of antibodies to various P. falciparum antigens. The data point to a remarkably similar response to malaria in the Mossi and Rimaibé, while the Fulani are clearly less parasitized, less affected by the disease, and more responsive to all antigens tested. No difference in the use of malaria protective measures was demonstrated that could account for these findings, and sociocultural or environmental factors do not seem to be involved. Known genetic factors of resistance to malaria did not show higher frequencies in the Fulani. The differences in the immune response were not explained by the entomological observations, which indicated substantially uniform exposure to infective bites. The available data support the existence of unknown genetic factors, possibly related to humoral immune responses, determining interethnic differences in the susceptibility to malaria.

Transcriptional regulation of the rat Müllerian inhibiting substance type II receptor in rodent Leydig cells

Müllerian inhibiting substance (MIS) causes regression of the fetal Müllerian duct on binding a heteromeric complex of types I and II cell-surface receptors in the fetal urogenital ridge. The MIS type II receptor (MISRII), which provides specificity for MIS, is also expressed in the adult testis, ovary, and uterus. The rat MISRII promoter was cloned to study the molecular mechanisms underlying its temporal and cell-specific expression. The 1.6-kilobase (kb) promoter contained no recognizable TATA or CAAT box, but there was a consensus Sp1 site upstream of the transcription initiation site. Two binding sites for the orphan nuclear receptor steroidogenic factor-1 (SF-1) are occupied in vitro by using nuclear extracts from R2C cells, an MIS-responsive rat Leydig cell line that expresses endogenous MISRII, with differing affinities, indicating that the distal SF-1 site is bound more avidly than is the proximal SF-1 site. R2C cells transfected with MISRII promoter/luciferase reporter constructs show a 12-fold induction with the 1.6-kb fragment and deletion of sequences upstream of −282-bp lowered luciferase expression to one-third. Mutation of both SF-1 sites greatly inhibited luciferase expression, whereas mutation of either site alone resulted in continuing activation by endogenous SF-1, indicating redundancy. In vitro binding and transcriptional analyses suggest that a proximal potential Smad-responsive element and an uncharacterized element also contribute to activation of the MISRII gene. R2C cells and MISRII promoter regulation can now be used to uncover endogenous transcription factors responsible for receptor expression or repression.

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

An alternative pathway for rod signals in the rodent retina: Rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.

Homeobox gene Prx3 expression in rodent brain and extraneural tissues

Different cDNA clones encoding a rat homeobox gene and the mouse homologue OG-12 were cloned from adult rat brain and mouse embryo mRNA, respectively. The predicted amino acid sequences of the proteins belong to the paired-related subfamily of homeodomain proteins (Prx homeodomains). Hence, the gene was named Prx3 and the mouse and rat genes are indicated as mPrx3 and rPrx3, respectively. In the mouse as well as in the rat, the predicted Prx3 proteins share the homeodomain but have three different N termini, a 12-aa residue variation in the C terminus, and contain a 14-aa residue motif common to a subset of homeodomain proteins, termed the “aristaless domain.” Genetic mapping of Prx3 in the mouse placed this gene on chromosome 3. In situ hybridization on whole mount 12.5-day-old mouse embryos and sections of rat embryos at 14.5 and 16.5 days postcoitum revealed marked neural expression in discrete regions in the lateral and medial geniculate complex, superior and inferior colliculus, the superficial gray layer of the superior colliculus, pontine reticular formation, and inferior olive. In rat and mouse embryos, nonneuronal structures around the oral cavity and in hip and shoulder regions also expressed the Prx3 gene. In the adult rat brain, Prx3 gene expression was restricted to thalamic, tectal, and brainstem structures that include relay nuclei of the visual and auditory systems as well as other ascending systems conveying somatosensory information. Prx3 may have a role in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation.

Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5′ flanking sequence (−855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5′ flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

α+-Thalassemia protects children against disease caused by other infections as well as malaria

In the South West Pacific region, the striking geographical correlation between the frequency of α+-thalassemia and the endemicity of Plasmodium falciparum suggests that this hemoglobinopathy provides a selective advantage against malaria. In Vanuatu, paradoxically, α+-thalassemia increases the incidence of contracting mild malaria in the first 2 years of life, but severe disease was too uncommon to assess adequately. Therefore, we undertook a prospective case-control study of children with severe malaria on the north coast of Papua New Guinea, where malaria transmission is intense and α+-thalassemia affects more than 90% of the population. Compared with normal children, the risk of having severe malaria was 0.40 (95% confidence interval 0.22–0.74) in α+-thalassemia homozygotes and 0.66 (0.37–1.20) in heterozygotes. Unexpectedly, the risk of hospital admission with infections other than malaria also was reduced to a similar degree in homozygous (0.36; 95% confidence interval 0.22–0.60) and heterozygous (0.63; 0.38–1.07) children. This clinical study demonstrates that a malaria resistance gene protects against disease caused by infections other than malaria. The mechanism of the remarkable protective effect of α+-thalassemia against severe childhood disease remains unclear but must encompass the clear interaction between this hemoglobinopathy and both malarial and nonmalarial infections.

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.