61 resultados para MALARIA VECTORS

em National Center for Biotechnology Information - NCBI


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Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd′ of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd′ suggests that the inversion may be the result of the transposable element’s activity. Characteristics of Odysseus’ terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.

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The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

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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.

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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.

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We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1α promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2–10 μg/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200–400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

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The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes.

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Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor α (TNF-α)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7–60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-α > Fas > perforin. TNF-α did not have a curative effect on infected hepatocytes, as the administration of TNF-α to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-α-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-α deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-α in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-α function have been removed from most Ad vectors, rendering them highly susceptible to TNF-α-mediated elimination.

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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.

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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.

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This paper deals with pattern recognition of the shape of the boundary of closed figures on the basis of a circular sequence of measurements taken on the boundary at equal intervals of a suitably chosen argument with an arbitrary starting point. A distance measure between two boundaries is defined in such a way that it has zero value when the associated sequences of measurements coincide by shifting the starting point of one of the sequences. Such a distance measure, which is invariant to the starting point of the sequence of measurements, is used in identification or discrimination by the shape of the boundary of a closed figure. The mean shape of a given set of closed figures is defined, and tests of significance of differences in mean shape between populations are proposed.

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Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.

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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.

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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.

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Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-α promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 × 1013 particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 μg/ml in SCID and over 400 μg/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.

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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.