Structure-based drug design studies of the interactions ofent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparumsarco/endoplasmic reticulum Ca2+-ATPase PfATP6

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosawere tested in silico against the Plasmodium falciparumCa2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP)-expressing Plasmodium bergheistrain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3) inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Immunoregulation in human malaria: the challenge of understanding asymptomatic infection

Asymptomatic Plasmodium infection carriers represent a major threat to malaria control worldwide as they are silent natural reservoirs and do not seek medical care. There are no standard criteria for asymptomaticPlasmodium infection; therefore, its diagnosis relies on the presence of the parasite during a specific period of symptomless infection. The antiparasitic immune response can result in reducedPlasmodium sp. load with control of disease manifestations, which leads to asymptomatic infection. Both the innate and adaptive immune responses seem to play major roles in asymptomatic Plasmodiuminfection; T regulatory cell activity (through the production of interleukin-10 and transforming growth factor-β) and B-cells (with a broad antibody response) both play prominent roles. Furthermore, molecules involved in the haem detoxification pathway (such as haptoglobin and haeme oxygenase-1) and iron metabolism (ferritin and activated c-Jun N-terminal kinase) have emerged in recent years as potential biomarkers and thus are helping to unravel the immune response underlying asymptomatic Plasmodium infection. The acquisition of large data sets and the use of robust statistical tools, including network analysis, associated with well-designed malaria studies will likely help elucidate the immune mechanisms responsible for asymptomatic infection.

Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum

The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum). Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9) in transgenic tomato plants from the T0 and T1 generations.

Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plasmodium spp.)

Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods. Considering the negative impact of avian malaria on penguins, the aim of this study was to detect the presence of Plasmodium spp. in Magellanic penguins using Polymerase Chain Reaction (PCR) and by verifying clinical, hematological, and biochemical alterations in blood samples as well as to verify the likely prognosis in response to infection. Blood samples were obtained from 75 penguins to determine packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), uric acid, total protein, albumin, globulin and aspartate aminotransferase (AST) activity levels. Whole blood samples were used for PCR assays. Plasmodium spp. was detected in 32.0% of the specimens using PCR and in 29.3% using microscopic analyses. Anorexia, diarrhea and neurological disorders were more frequent in penguins with malaria and a significant weight difference between infected and non-infected penguins was detected. PCV and MCV rates showed no significant difference. RBC and WBC counts were lower in animals with avian malaria and leukopenia was present in some penguins. Basophil and lymphocyte counts were lower in infected penguins along with high monocyte counts. There was no significant difference in AST activities between infected and non-infected animals. There was a significant increase in uric acid values, however a decrease in albumin values was observed in infected penguins. Based on this study, we concluded that Plasmodium spp. occurs in Magellanic penguins of rehabilitation centers in Southeastern Brazil, compromising the weight of infected animals with clinical alterations appearing in severe cases of this disease. It was also noted that, although the hematological abnormalities presented by these animals may not have been conclusive, leukopenia, monocytosis and the decrease of basophils and lymphocytes revealed an unfavorable prognosis, and Plasmodium spp. infections may progress with elevated uric acid concentration and low albumin levels.

Plasmodium spp. and Haemoproteus spp. infection in birds of the Brazilian Atlantic Forest detected by microscopy and polymerase chain reaction

In recent years haemosporidian infection by protozoa of the genus Plasmodium and Haemoproteus, has been considered one of the most important factors related to the extinction and/or population decline of several species of birds worldwide. In Brazil, despite the large avian biodiversity, few studies have been designed to detect this infection, especially among wild birds in captivity. Thus, the objective of this study was to analyze the prevalence of Plasmodium spp. and Haemoproteus spp. infection in wild birds in captivity in the Atlantic Forest of southeastern Brazil using microscopy and the polymerase chain reaction. Blood samples of 119 different species of birds kept in captivity at IBAMA during the period of July 2011 to July 2012 were collected. The parasite density was determined based only on readings of blood smears by light microscopy. The mean prevalence of Plasmodium spp. and Haemoproteus spp. infection obtained through the microscopic examination of blood smears and PCR were similar (83.19% and 81.3%, respectively), with Caracara plancus and Saltator similis being the most parasitized. The mean parasitemia determined by the microscopic counting of evolutionary forms of Plasmodium spp. and Haemoproteus spp. was 1.51%. The results obtained from this study reinforce the importance of the handling of captive birds, especially when they will be reintroduced into the wild.

Malaria vaccine: roadblocks and possible solutions

Malaria remains the most prevalent and devastating parasitic disease worldwide. Vaccination is considered to be an approach that will complement other strategies for prevention and control of the disease in the future. In the last 10 years, intense studies aimed at the development of a malaria vaccine have provided important knowledge of the nature of the host immunological mechanisms of protection and their respective target antigens. It became well established that protective immune responses can be generated against the distinct stages of Plasmodium. However, in general, protective immune responses are directed at stage-specific antigens. The elucidation of the primary structure of these antigens made possible the generation of synthetic and recombinant proteins that are being extensively used in experimental immunizations against the infection. Today, several epitopes of limited polymorphism have been described and protective immunity can be generated by immunization with them. These epitopes are being tested as primary candidates for a subunit vaccine against malaria. Here we critically review the major roadblocks for the development of a malaria vaccine and provide some insight on how these problems are being solved

Effects of autacoid inhibitors and of an antagonist on malaria infection in mice

The effects of p-chlorophenylalanine, an inhibitor of serotonin synthesis, indomethacin, an inhibitor of prostaglandin synthesis, cyproheptadine, a serotonin, bradykinin and histamine antagonist, were assessed separately and in combination with chloroquine (CQ) in Vom strains of Swiss albino mice (18-22 g) of either sex infected intraperitoneally with 1 x 10(7) Plasmodium yoelii nigeriensis-induced malaria. As prophylactic, these agents reduced from 31.9 ± 4.5 to 16.1 ± 8.1% the level of parasitemia relative to control but had no appreciable activity as curative agents when administered subcutaneously once daily for 4 days after 72 h of parasites innoculum in vivo. However, CQ alone and the combination of these agents with CQ in curative and prophylactic treatments significantly reduced (from 50.3 ± 5.8 to 4.9 ± 0.75%) the level of parasitemia (P < 0.05), which was taken only once 72 h after the parasites innoculum. The prophylactic result was shown to produce better results than the curative treatment. The data indicate that inhibitors and an antagonist can reduce the parasitemia load (the extent of damage and the severity of infection) as well as enhance the effects of CQ when combined with it for malaria therapy. The study reveals that the production of autacoids in established infection renders autacoid inhibitors and an antagonist ineffective for radical cure in malarial mice; however, selective inhibition of local hormones implicated in the pathological manifestations of malaria infection by autacoid inhibitors and an antagonist may be a possible pathway to reduce the severity of infection and the associated tissue damage and to enhance the efficacy of available anti-malarials.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes and the infected placenta: a two-way pathway

Malaria is undoubtedly the world's most devastating parasitic disease, affecting 300 to 500 million people every year. Some cases of Plasmodium falciparum infection progress to the deadly forms of the disease responsible for 1 to 3 million deaths annually. P. falciparum-infected erythrocytes adhere to host receptors in the deep microvasculature of several organs. The cytoadhesion of infected erythrocytes to placental syncytiotrophoblast receptors leads to pregnancy-associated malaria (PAM). This specific maternal-fetal syndrome causes maternal anemia, low birth weight and the death of 62,000 to 363,000 infants per year in sub-Saharan Africa, and thus has a poor outcome for both mother and fetus. However, PAM and non-PAM parasites have been shown to differ antigenically and genetically. After multiple pregnancies, women from different geographical areas develop adhesion-blocking antibodies that protect against placental parasitemia and clinical symptoms of PAM. The recent description of a new parasite ligand encoded by the var2CSA gene as the only gene up-regulated in PAM parasites renders the development of an anti-PAM vaccine more feasible. The search for a vaccine to prevent P. falciparum sequestration in the placenta by eliciting adhesion-blocking antibodies and a cellular immune response, and the development of new methods for evaluating such antibodies should be key priorities in mother-child health programs in areas of endemic malaria. This review summarizes the main molecular, immunological and physiopathological aspects of PAM, including findings related to new targets in the P. falciparum var gene family. Finally, we focus on a new methodology for mimicking cytoadhesion under blood flow conditions in human placental tissue.

Increased levels of glutamate in the central nervous system are associated with behavioral symptoms in experimental malaria

Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has been associated with cognitive, behavioral and motor dysfunctions, seizures and coma. The underlying mechanisms of CM are incompletely understood. Glutamate and other metabolites such as lactate have been implicated in its pathogenesis. In the present study, we investigated the involvement of glutamate in the behavioral symptoms of CM. Seventeen female C57BL/6 mice (20-25 g) aged 6-8 weeks were infected with P. berghei ANKA by the intraperitoneal route using a standardized inoculation of 10(6) parasitized red blood cells suspended in 0.2 mL PBS. Control animals (N = 17) received the same volume of PBS. Behavioral and neurological symptoms were analyzed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. Glutamate release was measured in the cerebral cortex and cerebrospinal fluid of infected and control mice by fluorimetric assay. All functional categories of the SHIRPA battery were significantly altered in the infected mice at 6 days post-infection (dpi) (P ≤ 0.05). In parallel to CM symptoms, we found a significant increase in glutamate levels in the cerebral cortex (mean ± SEM; control: 11.62 ± 0.90 nmol/mg protein; infected at 3 dpi: 10.36 ± 1.17 nmol/mg protein; infected at 6 dpi: 26.65 ± 0.73 nmol/mg protein; with EGTA, control: 5.60 ± 1.92 nmol/mg protein; infected at 3 dpi: 6.24 ± 1.87 nmol/mg protein; infected at 6 dpi: 14.14 ± 0.84 nmol/mg protein) and in the cerebrospinal fluid (control: 128 ± 51.23 pmol/mg protein; infected: 301.4 ± 22.52 pmol/mg protein) of infected mice (P ≤ 0.05). These findings suggest a role of glutamate in the central nervous system dysfunction found in CM.

Encontro do Plasmodium (S) tropiduri no Estado de São Paulo, Brasil

Relata-se o encontro, pela primeira vez no Estado de São Paulo - Brasil, de lagartos do gênero Tropidurus, parasitados pelo Plasmodium (S) tropiduri, mencionando alguns dos achados anteriores do parasito em outras áreas do país e na Venezuela. São salientadas as baixas parasitemias encontradas e a semelhança morfológica das formas estudadas com a descrição do parasita feita por Aragão e Neiva. São mostradas ainda as dificuldades no achado das formas exoeritrocíticas encontradas no trombócitos. Assinala-se o encontro em baixa densidade, do A. (N) evansae e A. (N.) argyritarsis, numa das áreas estudadas.

Biología de Anopheles (Kerteszia) neivai H., D. & K., 1913 (Diptera: Culicidae) en la Costa Pacifica de Colombia: IV - Estructura etárea y transmisión de malaria

Con el fin de determinar la dinamica de transmisión de malaria en el poblado de Charambirá (Chocó), Colombia, se estudió la estructura etárea de Anopheles neivai (reconocido vector en la Costa Pacífica) con base en su estado gonadotrófico. Se recolectaron mosquitos intradomiciliarmente durante el crepúsculo vespertino con cebos humanos y aspiradores bucales. Los mosquitos recolectados se mantuvieron en cajas cilíndricas de cartón con papel humedecido y dispensadores alimenticios hasta su disección al día siguiente. De los 200 mosquitos disecados entre septiembre y octubre de 1986,68 (34%) presentaban huellas de menos de tres oviposturas y los restantes (66%) habían efectuado al menos tres oviposturas. La diferencia entre el primer grupo considerado como mosquitos "no infectivos" y el segundo considerado como los "potencialmente infectivos" fue altamente significativa (X² = 10,68; P = 0,001). El 1,5% de A. neivai estudiados presentaban huellas correspondientes a 10 oviposturas mostrando una marcada longevidad y múltiples alimentaciones sanguíneas. Los resultados sugieren que existe un alto riesgo de contraer malaria en Charambirá durante el crepúsculo vespertino.

Populations of larvae of Anopheles spp. in natural breeding sites in Western Venezuela, an area of refractory malaria

Studies have been un dertaken into on the diversity and relative abundance of larvae of Anopheles (Nyssorhynchus) spp. in 22 permanent or temporary pools in an area of 70 km² in the eastern piedmont of the Venezuela Andes, between the mountains and the plains, an area in which malaria is refractory and A. nuñeztovari is present. Twelve species were identified, the most frequent, abundant and sympatric being A. triannulatus, A. albitarsis, A. nuñeztovari, A. oswaldoi and A. strodei. The samples from the permanent pools showed greater diversity of species and greater numbers of larvae than the samples from the temporary pools. The existence of the same larval associations in pools of other localities in the eastern piedmont of the Venezuelan Andes suggests the possibility of the making an ecological map of the breeding sites of A. nuñeztovari and for these anophelines in a region extending for 430 km.

A mathematical model for malaria transmission relating global warming and local socioeconomic conditions

OBJECTIVE: Sensitivity analysis was applied to a mathematical model describing malaria transmission relating global warming and local socioeconomic conditions. METHODS: A previous compartment model was proposed to describe the overall transmission of malaria. This model was built up on several parameters and the prevalence of malaria in a community was characterized by the values assigned to them. To assess the control efforts, the model parameters can vary on broad intervals. RESULTS: By performing the sensitivity analysis on equilibrium points, which represent the level of malaria infection in a community, the different possible scenarios are obtained when the parameters are changed. CONCLUSIONS: Depending on malaria risk, the efforts to control its transmission can be guided by a subset of parameters used in the mathematical model.