Low Fetal Weight is Directly Caused by Sequestration of Parasites and Indirectly by IL-17 and IL-10 Imbalance in the Placenta of Pregnant Mice with Malaria

The sequestration of infected erythrocytes in the placenta can activate the syncytiotrophoblast to release cytokines that affect the micro-environment and influence the delivery of nutrients and oxygen to fetus. The high level of IL-10 has been reported in the intervillous space and could prevent the pathological effects. There is still no data of Th17 involvement in the pathogenesis of placental malaria. This study was conducted to reveal the influence of placental IL-17 and IL-10 levels on fetal weights in malaria placenta. Seventeen pregnant BALB/C mice were divided into control (8 pregnant mice) and treatment group (9 pregnant mice infected by Plasmodium berghei). Placental specimens stained with hematoxylin and eosin were examined to determine the level of cytoadherence by counting the infected erythrocytes in the intervillous space of placenta. Levels of IL-17 and IL-10 in the placenta were measured using ELISA. All fetuses were weighed by analytical balance. Statistical analysis using Structural Equation Modeling showed that cytoadherence caused an increased level of placental IL-17 and a decreased level of placental IL-10. Cytoadherence also caused low fetal weight. The increased level of placental IL-17 caused low fetal weight, and interestingly low fetal weight was caused by a decrease of placental IL-10. It can be concluded that low fetal weight in placental malaria is directly caused by sequestration of the parasites and indirectly by the local imbalance of IL-17 and IL-10 levels.

Anopheles gambiae eicosanoids modulate Plasmodium berghei survival from oocyst to salivary gland invasion

Eicosanoids affect the immunity of several pathogen/insect models, but their role on the Anopheles gambiae response to Plasmodium is still unknown. Plasmodium berghei-infected mosquitoes were injected with an eicosanoid biosynthesis inhibitor, indomethacin (IN), or a substrate, arachidonic acid (AA), at day 7 or day 12 post-infection (p.i.). Salivary gland invasion was evaluated by sporozoite counts at day 21 p.i. IN promoted infection upon sporozoite release from oocysts, but inhibited infection when sporozoites were still maturing within the oocysts, as observed by a reduction in the number of sporozoites reaching the salivary glands. AA treatment had the opposite effect. We show for the first time that An. gambiae can modulate parasite survival through eicosanoids by exerting an antagonistic or agonistic effect on the parasite, depending on its stage of development.

Modulation of the oxidative stress in malaria infection by clotrimazole

Antimycotic clotrimazole (CTZ) has demonstrated remarkable activity against Plasmodium falciparum in vitro and in vivo. Hemoglobin degradation by Plasmodium parasites makes amino acids available for protein synthesis, inducing oxidative stress in infected cells and producing free heme. These events represent biochemical targets for potential antimalarials. In this study, we have tested the ability of CTZ to modify the oxidative status in Plasmodium berghei-infected erythrocytes. After hemolysis, activities of superoxide dismutase (SOD), catalase (CAT), glutathione cycle and NADPH+H+-producing dehydrogenases were investigated using UV-visible spectrophotometry. Thiobarbituric acid reactive substances (TBARS) were evaluated as a marker of lipid damage. Results showed that CTZ significantly decreased the overall activity of 6-phosphagluconate dehydrogenase (6PGD) compared to infected and non-treated cells; consequently, the glutathione cycle was inhibited, leaving the parasite vulnerable to the oxidative stress originating from hemoglobin degradation. As a compensatory response, CTZ prevented some loss of SOD and CAT activities in infected cells. The infection triggered lipid peroxidation in erythrocytes, which was decreased by CTZ. These results suggest the presence of a redox unbalance in cells treated with CTZ, discussing a possible effect of this compound disturbing the oxidative status in a Plasmodium berghei-infection.

O antifúngico clotrimazol (CTZ) tem demonstrado notável atividade contra Plasmodium falciparum. A degradação da hemoglobina por Plasmodium para a obtenção dos aminoácidos necessários à síntese protéica induz estresse oxidativo em eritrócitos devido à liberação de hemos oxidantes. Estes eventos representam alvos bioquímicos para a produção de antimaláricos potenciais. Neste estudo, testamos a capacidade do CTZ para modificar o estado oxidativo em eritrócitos infectados com Plasmodium berghei. Depois da hemólise, as atividades da superóxido dismutase (SOD), catalase (CAT), desidrogenases produtoras de NADPH+H+ e do ciclo de glutationa (GSH) foram investigados. A produção das espécies reativas ao ácido tiobarbitúrico (TBARS) foi avaliada como marcador de dano lipídico. Os resultados mostraram que o CTZ diminuiu a atividade da 6-fosfogliconato desidrogenase (6PGD), em comparação com eritrócitos infectados e não tratados. Consequentemente, o ciclo da GSH foi inibido, tornando os parasitas vulneráveis ao estresse oxidativo resultante da degradação da hemoglobina. Como resposta compensatória, CTZ impediu a perda de atividade da SOD e CAT nas células infectadas. A infecção induz peroxidação lipídica nos eritrócitos, sendo esta diminuída pelo CTZ. Estes resultados sugerem a existência de desequilíbrio redox nas células tratadas com CTZ, interferindo, assim, com o estado oxidativo verificado durante a infecção malárica.

Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics / Alteración en el metabolismo de la hemoglobina y actividad antimalárica in vivo de azoles antimicóticos

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ß-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ß-hematin synthesis (IßHS) and hemoglobin proteolysis (IHbP) in vitro. The ß-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5 percent, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 µM and 14.4 ± 1.4 µM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0 percent and 55.3 ± 3.6 percent, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia ( percentP) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0 percent compared to 26.6 ± 3.7 percent, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.

Los parásitos del género Plasmodium degradan la hemoglobina hospedera obteniendo aminoácidos libres para su síntesis proteica. Durante este evento, unidades de hemo libre tóxicas cristalizan espontáneamente formando un pigmento no tóxico denominado ß-hematina. En este trabajo, se investigó la capacidad de un grupo de azoles antimicóticos: clotrimazol (CTZ), ketoconazol (KTZ) y fluconazol (FCZ), en inhibir la síntesis de ß-hematina y la proteólisis de la globina. La síntesis de ß-hematina se registro por espectrofotometría a 405 nm y la proteólisis de la hemoglobina se determino por SDS-PAGE 15 por ciento seguido por análisis densitométrico de las bandas de hemoglobina intactas. Los compuestos fueron también ensayados in vivo en un modelo de malaria murina. CTZ y KTZ inhibieron la síntesis de ß-hematina con CI50 entre 10 y 15 µM y bloquearon la proteólisis de la hemoglobina (80.01 ± 2.04 por ciento y 55.33 ± 3.57 por ciento, respectivamente). En relación directa con los resultados encontrados in vitro, el CTZ redujo la parasitemia de ratones infectados en forma significativa, así como prolongó lo días de sobrevivencia post-infección en comparación con animales controles no tratados. Se sugiere así que la inhibición del metabolismo de la hemoglobina por los antimicóticos azólicos pudiera ser el mecanismo responsable de su actividad antimalárica.

Plasmodium berghei ANKA infection induces thymocyte apoptosis and thymocyte depletion in CBA mice

Immune responses to malaria infections are characterized by strong T and B cell activation, which, in addition of potentially causing immunopathology, are of poor efficacy against the infection. It is possible that the thymus is involved in the origin of immunopathological reactions and a target during malaria infections. This work was developed in an attempt to further clarify these points. We studied the sequential changes in the thymus of CBA mice infected with Plasmodium berghei ANKA, a model in which 60-90 percent of the infected animals develop cerebral malaria. During the acute phase of infection, different degrees of thymocyte apoptosis were recorded: (1) starry-sky pattern of diffuse apoptosis with maintenance of cortical-medullary structure; (2) intense apoptosis with cortical atrophy, with absence of large cells; (3) severe cortical thymocyte depletion, resulting in cortical-medullary inversion. In the latter, only residual clusters of small thymocytes were observed within the framework of epithelial cells. The intensity of thymus alterations could not be associated with the degree of parasitemia, the expression of clinical signs of cerebral malaria or intensity of brain lesions. The implications of these events for malaria immunity and pathology are discussed.

Estudo sobre a possível atividade terapêutica da deferoxamina na infecçäo experimental de camundongos pelo Plasmodium berghei / The possible therapeutic activity of deferoxamine in experimental infections of mice by Plasmodium berghei

A deferoxamina é substância química dotada de propriedades quelantes relativas aos sais de ferro. A depleçäo desse mineral no organismo poderia influir, segundo algumas opiniöes, sobre o metabolismo de plasmódios que se desenvolvem no interior das hemácias. Para analisar essa possibilidade administramos doses cotidianas de 300 ou 1.000 mg/Kg do composto, durante cinco e 15 dias, a camundongos infectados pelo Plasmodium berghei. A mortalidade dos animais e a contagem do número de parasitas no sangue foram os parâmetros utilizados para verificar a atividade da deferoxamina. Os resultados evidenciaram aumento gradual e progressivo da mortalidade e da parasitemia em todos os roedores, sem diferenças constatadas nos controles. Assim, pelo menos de acordo com o estudo efetuado, dependente de modelo experimental, a substância näo se mostrou promissora para o tratamento da malária

In vitro culture of erythrocytic stages, including gametocytes of Plasmodium berghei (NK 65 strain) using candle jar method and a newly designed continuous medium flow apparatus.

Three methods have been described for cultivation of erythrocytic stages, specially gametocytes of P. berghei NK65 strain, (1) by using vial candle jar where the cultures were subcultured by addition of fresh erythrocytes, (2) a newly designed simple and compact continuous medium flow apparatus, where medium was continuously perfused but fresh erythrocytes were not added and (3) where the subcultures were also done using simple and compact continuous medium flow apparatus for comparison. The maximum percentage of parasitized erythrocytes obtained by these methods was 24.3, 27.1 and 26.4% respectively. Parasites in vials could survive for more than 10 days with 3 to 4 subcultures with maximum 1.96% gametocytaemia. However, the gametocytaemia in continuous medium flow apparatus, where subcultures were not made reached 2.2% compared to that of 2.7% in this apparatus where subcultures were done. The asexual as well as sexual stages of this parasite survived for about 16-18 days in compact continuous medium flow apparatus, where at least 7 subcultures were done.

Ontogeny of ookinete of Plasmodium berghei (NK 65): a scanning electron microscopic study.

The ontogeny of ookinete of Plasmodium berghei (NK 65) was studied in vector Anopheles stephensi fed on infected Mastomys natalensis. The round zygote transformed into an ookinete after passing through following stages-1 gram-seed shaped zygote, 2 comma-shaped stage, 3 semilunar and 4 banana shaped ookinete. Each fully formed ookinete had a 'Conule' at the anterior end of the body. In some ookinetes under SEM a depression was observed in the posterior half of the body. The function of the depression was not known.