Primaquine-thiazolidinones block malaria transmission and development of the liver exoerythrocytic forms.

BACKGROUND: Primaquine is an anti-malarial used to prevent Plasmodium vivax relapses and malaria transmission. However, PQ metabolites cause haemolysis in patients deficient in the enzyme glucose-6-phosphate dehydrogenase (G6PD). Fifteen PQ-thiazolidinone derivatives, synthesized through one-post reactions from primaquine, arenealdehydes and mercaptoacetic acid, were evaluated in parallel in several biological assays, including ability to block malaria transmission to mosquitoes. RESULTS: All primaquine derivatives (PQ-TZs) exhibited lower cell toxicity than primaquine; none caused haemolysis to normal or G6PD-deficient human erythrocytes in vitro. Sera from mice pretreated with the test compounds thus assumed to have drug metabolites, caused no in vitro haemolysis of human erythrocytes, whereas sera from mice pretreated with primaquine did cause haemolysis. The ability of the PQ-TZs to block malaria transmission was evaluated based on the oocyst production and percentage of mosquitoes infected after a blood meal in drug pre-treated animals with experimental malaria caused by either Plasmodium gallinaceum or Plasmodium berghei; four and five PQ-TZs significantly inhibited sporogony in avian and in rodent malaria, respectively. Selected PQ-TZs were tested for their inhibitory activity on P. berghei liver stage development, in mice and in vitro, one compound (4m) caused a 3-day delay in the malaria pre-patent period. CONCLUSIONS: The compound 4m was the most promising, blocking malaria transmissions and reducing the number of exoerythrocytic forms of P. berghei (EEFs) in hepatoma cells in vitro and in mice in vivo. The same compound also caused a 3-day delay in the malaria pre-patent period.

Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes.

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d-amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so-called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all-D-AII and all-D-VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro-AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a ß-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a ß-turn conformation, while retro-AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro-AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro-AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation.

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development.

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites.

Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development.

The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein-coding sequence to inactivate the enzyme while maintaining the protein's structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut-specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2.

A expressão de WARP, um alvo potencial para Vacinas de Bloqueio de Transmissão, durante o desenvolvimento sexual de Plasmodium gallinaceum

Durante o ciclo de vida dos parasitos causadores da malária, uma das fases cruciais é o desenvolvimento sexual e a conseqüente invasão do epitélio intestinal do hospedeiro invertebrado. Proteínas dos estágios do ciclo esporogônico podem ser alvos para vacinas Bloqueadoras de Transmissão (TBVs). Uma das proteínas micronemais que já demonstrou ser um alvo promissor para a inibição do desenvolvimento de oocistos e, portanto, da transmissão, é a WARP (von Willebrand Factor A Domain Related Protein), uma proteína secretada, aparentemente envolvida com a adesão e locomoção dos parasitos. Estudos recentes demonstraram que WARP é sujeita à repressão traducional em macrogametócitos de P. berghei, com o seu mRNA sendo silenciado pela proteína DOZI através da formação de uma ribonucleoproteína. Os objetivos deste estudo foram i) Analisar o perfil de expressão da proteína WARP nos estágios sexuais de P. gallinaceum, ii) Comparar o perfil de transcrição de WARP, durante o desenvolvimento sexual, com perfis de alguns genes relacionados à regulação da expressão ou à invasão epitelial, e iii) Avaliar o potencial da proteína WARP como um candidato à vacina através da análise do seu padrão de expressão.

O domínio vWA de PfWARP foi produzido como uma proteína recombinante que foi usada para a produção de anticorpos policlonais. Foram realizados experimentos de microscopia confocal usando estes anticorpos para se detectar WARP em estágios sexuais cultivados de P. gallinaceum. RT-PCR foi usada para detectar WARP e os outros genes estudados a partir de amostras de sangue contendo gametócitos e a partir de intestinos de Aedes fluviatilis infectados, coletados até 24 horas após a alimentação. A proteína WARP pode ser detectada desde os estágios iniciais do desenvolvimento de oocinetos, em zigotos recém-fertilizados, até as formas maduras alongadas. Em zigotos, sua expressão parece ser intra-vesicular e localizada na periferia citoplasmática, próxima à membrana celular, e em oocinetos maduros, WARP apresenta uma distribuição granular com concentração focal na região apical, corroborando sua localização micronemal. Seu transcrito foi detectado, por RT-PCR, em gametócitos de P. gallinaceum. Ainda, o transcrito para a proteína PgDOZI também foi detectado em gametócitos, indicando que a repressão traducional possa ser um mecanismo de regulação gênica também nesta espécie, e que WARP seja, provavelmente, um de seus alvos de regulação. Esta é a primeira vez que a proteína WARP é detectada tão prematuramente durante o desenvolvimento de oocinetos e a primeira evidência de expressão dos genes DOZI, α-Tubulina e MAOP em P. gallinaceum.

A expressão de WARP, um alvo potencial para Vacinas de Bloqueio de Transmissão, durante o desenvolvimento sexual de Plasmodium gallinaceum / The Warp of expression, an potential target to vaccines of transmission of blockade, during the sexual development of Plasmodium gallinaceum

Durante o ciclo de vida dos parasitos causadores da malária, uma das fases cruciais é o desenvolvimento sexual e a conseqüente invasão do epitélio intestinal do hospedeiro invertebrado. Proteínas dos estágios do ciclo esporogônico podem ser alvos para vacinas Bloqueadoras de Transmissão (TBVs). Uma das proteínas micronemais que já demonstrou ser um alvo promissor para a inibição do desenvolvimento de oocistos e, portanto, da transmissão, é a WARP (von Willebrand Factor A Domain Related Protein), uma proteína secretada, aparentemente envolvida com a adesão e locomoção dos parasitos. Estudos recentes demonstraram que WARP é sujeita à repressão traducional em macrogametócitos de P. berghei, com o seu mRNA sendo silenciado pela proteína DOZI através da formação de uma ribonucleoproteína. Os objetivos deste estudo foram i) Analisar o perfil de expressão da proteína WARP nos estágios sexuais de P. gallinaceum, ii) Comparar o perfil de transcrição de WARP, durante o desenvolvimento sexual, com perfis de alguns genes relacionados à regulação da expressão ou à invasão epitelial, e iii) Avaliar o potencial da proteína WARP como um candidato à vacina através da análise do seu padrão de expressão.

O domínio vWA de PfWARP foi produzido como uma proteína recombinante que foi usada para a produção de anticorpos policlonais. Foram realizados experimentos de microscopia confocal usando estes anticorpos para se detectar WARP em estágios sexuais cultivados de P. gallinaceum. RT-PCR foi usada para detectar WARP e os outros genes estudados a partir de amostras de sangue contendo gametócitos e a partir de intestinos de Aedes fluviatilis infectados, coletados até 24 horas após a alimentação. A proteína WARP pode ser detectada desde os estágios iniciais do desenvolvimento de oocinetos, em zigotos recém-fertilizados, até as formas maduras alongadas. Em zigotos, sua expressão parece ser intra-vesicular e localizada na periferia citoplasmática, próxima à membrana celular, e em oocinetos maduros, WARP apresenta uma distribuição granular com concentração focal na região apical, corroborando sua localização micronemal. Seu transcrito foi detectado, por RT-PCR, em gametócitos de P. gallinaceum. Ainda, o transcrito para a proteína PgDOZI também foi detectado em gametócitos, indicando que a repressão traducional possa ser um mecanismo de regulação gênica também nesta espécie, e que WARP seja, provavelmente, um de seus alvos de regulação. Esta é a primeira vez que a proteína WARP é detectada tão prematuramente durante o desenvolvimento de oocinetos e a primeira evidência de expressão dos genes DOZI, α-Tubulina e MAOP em P. gallinaceum.

Exoerythrocytic development of Plasmodium gallinaceum sporozoites in a chicken fibroblast cell line and inhibition of the cell invasion by specific anti-sporozoite monoclonal antibodies.

Cultivation of the Plasmodium gallinaceum exoerythrocytic forms from sporozoites was attempted in three different cell lines: HEPG2-A16 (from a human hepatoma), VERO (monkey kidney epithelial cells) and SL-29 (chicken embryo fibroblast cells). The sporozoites invaded all three cells types but their development into exoerythrocytic forms occurred only in the SL-29 cells. In the presence of specific monoclonal antibodies against the major circumsporozoite protein, there were varying degrees of inhibition of parasite invasion of the SL-29 cells. Of seven monoclonal antibodies tested, two completely inhibited cell invasion at high concentrations and caused intense inhibition at concentrations as low as 2.5 micrograms/ml, four caused intense inhibition at these various concentrations, and one had no effect on sporozoite invasion.

A method for screening drugs against the liver stages of malaria using Plasmodium gallinaceum and Aedes mosquitos.

1. The radical cure of human malaria caused by Plasmodium vivax requires two drugs, i.e., a blood schizontocide such as chloroquine to clear the circulating parasites, and primaquine aimed at the liver stages (hyponozoites) responsible for the late relapses of this parasite. Primaquine is unique as a radical curative drug but is highly toxic. The only useful model currently available for screening drugs to replace primaquine is Plasmodium cynomolgi-induced malaria in Rhesus monkeys. Because of the limited availability and cost of these animals, the development of non-primate models for such screening would be of considerable value. 2. We used a drug-screening assay for the liver stage malaria parasite based on the ability of such drugs to stop development of gametocytes in the mosquito vector. The inhibition of the sporogonic cycle of malaria in the mosquito by primaquine (15 mg/kg) was confirmed here and used for re-evaluation of the gametocyte method. 3. We observed that the level of parasitemia in the untreated control chicken used to infect mosquitos was a crucial factor affecting the subsequent development of sporogony. Thus, parasitemia was carefully controlled in the studies involving oocyst development. Parasitemias lower than 6% at the beginning of the experiment and increasing were found to be most appropriate for the production of the infectious gametocytes during a period of 8 h.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro development of exoerythrocytic forms of Plasmodium gallinaceum sporozoites in avian macrophages.

Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. The host cells were macrophage precursors from chicken bone marrow. At various times after introduction of sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). The time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until the end of the primary exoerythrocytic schizogony.

The immunodominant surface antigen of Plasmodium gallinaceum is present in both the salivary gland and oocyst sporozoites.

1. Monoclonal antibodies (MAbs) against surface antigens of Plasmodium gallinaceum sporozoites, an avian malaria parasite, were produced using spleen cells from mice immunized with sporozoites from mosquito salivary glands (SGS) or from midguts containing oocysts (OoS). 2. All of the 15 MAbs tested (11 anti-SGS and 4 anti-OoS) reacted with SGS and OoS by indirect immunofluorescence and circumsporozoite precipitation reactions. Fourteen of these MAbs (11 anti-SGS and 3 anti-OoS) produced a Western blot (WB) pattern identical to that produced with serum from mice hyperimmunized with viable intact sporozoites. 3. All MAbs and the immune sera recognized only two polypeptide bands of approximate molecular weight 76 and 64 kDa. 4. No difference in the WB pattern was observed when 9- or 12-day SGS or OoS extracts were used as antigens in WB. This antigenic similarity was confirmed when the total protein extracts were visualized on silver-stained SDS-PAGE gel.

Experimental vaccination of chicks with Plasmodium gallinaceum sporozoites. I. Circumsporozoite proteins are expressed by sporozoites recovered from both salivary glands and midguts of mosquitoes.

Immunogenicity of Plasmodium gallinaceum sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8-9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal.(ABSTRACT TRUNCATED AT 250 WORDS)