Falciparum NF54 Research Articles

Exitoso cultivo in vitro de gametocitos de Plasmodium falciparum

The sexual stages of Plasmodium falciparum have not been studied in as much detail as the asexual stages due to the lack of standardized in vitro cultures as well as difficulties in identifying the sexual development stages of the parasite. These difficulties hamper the studies on biology, metabolism, gene expression and protein synthesis during sexual stages. Each of these facets are important targets in antimalarial drug research, particularly the identification of potential therapeutic agents against Plasmodium (derived mainly from plants). An in vitro culture of P. falciparum gametocytes was established to standardize the identification of its five developmental stages and ensure their continuous production. The in vitro gametocyte culture was established from the P. falciparum NF54 strain in RPMI culture medium, with assessment of the asexual and sexual parasitaemia. The medium was supplemented with type A Rh+ red blood cells only on the first day of culture. Subsequently, the medium was changed daily, together with addition of gas mixture (90% N2, 5% O2, 5% CO2) and maintenance of the culture temperature at 37 degrees C. When asexual parasitaemia reached 3 to 5%, the medium was changed by doubling its volume. We standardized an in vitro culture for sexual stages of P. falciparum that can be used for future studies about evaluation of compounds of synthetic or natural origen against the sexual stage, which may permit to develop new control strategies against malaria.

Modification of eukaryotic initiation factor 5A from Plasmodium vivax by a truncated deoxyhypusine synthase from Plasmodium falciparum: An enzyme with dual enzymatic properties

The increasing resistance of the malaria parasites enforces alternative directions in finding new drug targets. Present findings from the malaria parasite Plasmodium vivax, causing tertiary malaria, suggest eukaryotic initiation factor 5A (eIF-5A) to be a promising target for the treatment of malaria. Previously we presented the 162 amino acid sequence of eukaryotic initiation factor 5A (eIF-5A) from Plasmodium vivax. In the present study, we have expressed and purified the 20 kDa protein performed by one-step Nickel chelate chromatography. In Western blot experiments eIF-5A from P. vivax crossreacts with a polyclonal anti-eIF-5A antiserum from the plant Nicotiana plumbaginifolia ( Solanaceae). Transcription of eIF-5A can be observed in both different developmental stages of the parasite being prominent in trophozoites. We recently published the nucleic acid sequence from a genomic clone of P. falciparum strain NF54 encoding a putative deoxyhypusine synthase (DHS), an enzyme that catalyzes the post-translational modification of eIF-5A. After removal of 22 amino acids DHS was expressed as a Histidin fusion protein and purified by Nickel affinity chromatography. Truncated DHS from P. falciparum modifies eIF-5A from P. vivax. DHS from P. falciparum NF54 is a bi-functional protein with dual enzymatic specificities, that is, DHS activity and homospermidine synthase activity (HSS) (0.047 pkatal/mg protein) like in other eukaryotes. Inhibition of DHS from P. falciparum resulted in a K i of 0.1 μM for the inhibitor GC7 being 2000-fold less than the nonguanylated derivative 1,7-diaminoheptane. Dhs transcription occurs in both develomental stages suggesting its necessity in cell proliferation.

Synthesis of 2‐[3,5‐Substituted Pyrazol‐1‐yl]‐4,6‐trisubstituted Triazine Derivatives as Antimalarial Agents.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of 2-[3,5-substituted pyrazol-1-yl]-4,6-trisubstituted triazine derivatives as antimalarial agents

A series of 22 compounds were synthesized and screened against Plasmodium falciparum NF-54 strain. Of the screened compounds, 6 compounds showed MIC in the range between 1 and 2 μg/mL. These compounds are 32 times more potent than the cycloguanil which was used as the standard drug.

An Immunologically Cryptic Epitope of Plasmodium falciparum Circumsporozoite Protein Facilitates Liver Cell Recognition and Induces Protective Antibodies That Block Liver Cell Invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.

A short synthesis and biological evaluation of potent and nontoxic antimalarial bridged bicyclic beta-sulfonyl-endoperoxides.

The syntheses and in vitro antimalarial screening of 50 bridged, bicyclic endoperoxides of types 9-13 are reported. In contrast to antimalarial trioxanes of the artemisinin family, but like yingzhaosu A and arteflene, the peroxide function of compounds 9-13 is contained in a 2,3-dioxabicyclo[3.3.1]nonane system 6. Peroxides 9 and 10 (R(1) = OH) are readily available through a multicomponent, sequential, free-radical reaction involving thiol-monoterpenes co-oxygenation (a TOCO reaction). beta-Sulfenyl peroxides 9 and 10 (R(1) = OH) are converted into beta-sulfinyl and beta-sulfonyl peroxides of types 11-13 by controlled S-oxidation and manipulation of the tert-hydroxyl group through acylation, alkylation, or dehydration followed by selective hydrogenation. Ten enantiopure beta-sulfonyl peroxides of types 12 and 13 exhibit in vitro antimalarial activity comparable to that of artemisinin (IC(50) = 6-24 nM against Plasmodium falciparum NF54). In vivo testing of a few selected peroxides against Plasmodium berghei N indicates that the antimalarial efficacies of beta-sulfonyl peroxides 39a, 46a, 46b, and 50a are comparable to those of some of the best antimalarial drugs and are higher than artemisinin against chloroquine-resistant Plasmodium yoelii ssp. NS. In view of the nontoxicity of beta-sulfonyl peroxides 39a, 46a, and 46b in mice, at high dosing, these compounds are regarded as promising antimalarial drug candidates.

Evaluation of selected Sudanese medicinal plants for their in vitro activity against hemoflagellates, selected bacteria, HIV-1-RT and tyrosine kinase inhibitory, and for cytotoxicity

Ethnobotanical investigations led to the selection of 19 plant species, used traditionally in Sudan against malaria and other similar tropical diseases, for further studies. Pamianthe peruviana (Amaryllidaceae) exhibited significant activity against a chloroquine-resistant Plasmodium falciparum strain (K1) and a chloroquine-sensitive strain (NF54) with IC 50 values of 0.6 and 1.1 μg/ml, respectively. Additionally, P. peruviana showed considerable activities against Trypanosoma brucei rhodesiense (IC 50 1.5 μg/ml) and T. cruzi (IC 50 11.8 μg/ml). The antiplasmodial activity of the different extracts of Salvadora persica (Salvadoraceae) against P. falciparum NF54 strain were found to be 0.6 μg/ml (stems) and 0.7 μg/ml (leaves). Extracts of different parts of Combretum hartmannianum (Combretaceae) possessed significant activity against the chloroquine-sensitive P. falciparum strain (NF54) with IC 50 values of 0.2 μg/ml (bark), 0.4 μg/ml (stem) and 4.3 μg/ml (leaves). Most interestingly, the extracts of the leaves of C. hartmannianum totally inhibited the enzyme HIV-1 reverse transcriptase (HIV-1 RT) at a concentration of 66 μg/ml. A comparably strong activity against p56 lck tyrosine kinase was also seen for this extract.

Rare Sesquiterpenes from the Algicolous Fungus Drechslera dematioidea

From the inner tissue of the marine red alga Liagora viscida the fungus Drechslera dematioidea was isolated. After mass cultivation, the fungus was investigated for its secondary metabolite content, and 10 new sesquiterpenoids [isosativenetriol (1), drechslerines A (2) and B (3), 9-hydroxyhelminthosporol (5), drechslerines C-G (6-10), and sativene epoxide (12)] were isolated. Compounds 8 and 10 exhibited antiplasmodial activity against Plasmodium falciparum strains K1 and NF54. The known compounds helminthosporol (4), cis-sativenediol (11), isocochlioquinone A (14), isocochlioquinone C (15), and cochlioquinone B (16) were also isolated. All structures were elucidated using spectroscopic methods, mainly 1D and 2D NMR and MS.

In vitro activity of aurones against Plasmodium falciparum strains K1 and NF54.

A series of naturally occurring aurones (e. g., Rubiaceae, Cyperaceae) was synthesized and tested for the ability to inhibit erythrocytic stages of Plasmodium falciparum strains in vitro. Some of the these compounds exhibited antiplasmodial activity in the micromol range, determined as fifty percent-inhibitory concentrations (IC(50)). Drug activity was not associated with cytotoxicity for the mammalian tumor cell lines KB and SKMel (IC(50) > 3.0 microM). The most active compound was 4,6,4'-triacetyl-3',5'-dimethoxy-2-aurone with IC(50) values of 0.007 microM and 0.18 microM for the P. falciparum strains K1 and NF54, respectively. Interestingly, the multiple drug-resistant P. falciparum strain K1 was more sensitive to tested aurones than the drug-susceptible strain NF54.

Antimalarial and cytotoxic potential of four quassinoids from Hannoa chlorantha and Hannoa klaineana, and their structure-activity relationships

Hannoa chlorantha and Hannoa klaineana (Simaroubaceae) are used in traditional medicine of Central African countries against fevers and malaria. Four stem bark extracts from H. klaineana and four quassinoids from H. chlorantha were examined in vitro against Plasmodium falciparum NF 54. The extracts displayed good activities, while the quassinoids were highly active, with IC 50 values well below 1 μgml −1, those of chaparrinone and 15-desacetylundulatone being much lower than 0.1 μgml −1 (0.037 and 0.047 μgml −1, respectively). Chaparrinone is five times more active than 14-hydroxychaparrinone against P. falciparum, indicating that the hydroxyl function at C-14 is unfavourable for antiplasmodial activity. As 14-hydroxychaparrinone has a seven-times higher cytotoxic activity against P-388 cells than chaparrinone, the latter compound has the better antiplasmodial therapeutic index. All four quassinoids were evaluated in vivo in a standard 4-day test as well. 15-Desacetylundulatone was proven to be the most active compound, almost totally suppressing the parasitaemias of OF1 mice for at least 7 days, while both chaparrinone and 14-hydroxychaparrinone were active for at least 4 days. Quassinoids have ED 50 values much lower than 50 mgkg −1 body weight day −1 and none of them caused obvious side effects. The keto function at C-2 in 15-desacetylundulatone is apparently of crucial importance for its high activity. 6-α-Tigloyloxyglaucarubol was not active at all. Chaparrinone is considered the most interesting of the investigated quassinoids and its in-vivo antimalarial potential will be examined further.

Regular production of infective sporozoites of Plasmodium falciparum and P. vivax in laboratory-bred Anopheles albimanus.

One of the major constraints for studies on the sporogonic cycle of the parasites causing human malaria, and on the protective efficacy of pre-erythrocytic vaccines, is the scarcity of laboratory-reared Anopheles mosquitoes as a source of infective sporozoites. The aim of the present study was to reproduce the life-cycles of Plasmodium falciparum and P. vivax in the laboratory and so develop the ability to produce infective sporozoites of these two species regularly under laboratory conditions. Colonized Anopheles albimanus, of Buenaventura and Tecojate strains, were infected by feeding either on Plasmodium-infected blood, from human patients or experimentally inoculated Aotus monkeys, or on gametocytes of the P. falciparum NF-54 isolate grown in vitro. The monkeys were infected with the blood stages of a Colombian P. vivax isolate and then, after recovery, with the Santa Lucia strain of P. falciparum from El Salvador. Although both of the mosquito strains used were successfully infected with both parasite species, the Buenaventura strain of mosquito was generally more susceptible to infection than the Tecojate strain, and particularly to infection with the parasites from the patients, who lived where this strain of mosquitoes was originally isolated. Monkeys injected intravenously with the P. vivax sporozoites produced in the mosquitoes developed patent sexual and asexual parasitaemias; the gametocytes that developed could then be used to infect mosquitoes, allowing the development of more sporozoites. However, experimental infections failed to establish after the P. falciparum sporozoites were used to inoculate monkeys. The ability to reproduce the complete life cycle of P. vivax in the laboratory, from human to mosquito and then to monkey, should greatly facilitate many studies on vivax malaria and on the efficacy of candidate malaria vaccines. The availability of the sporogonic cycles of P. falciparum from three different sources should also permit a variety of biological studies.

ELISA Method for Detecting Anti-Plasmodium relictum and Anti-Plasmodium elongatum Antibody in Infected Duckling Sera Using Plasmodium falciparum Antigens

An enzyme-linked immunosorbent assay (ELISA) with 3 Plasmodium falciparum NF-54 antigens, R32tet32, P.F.R27, and crude red blood cell extract (CRBCE), was tested for detection of anti-Plasmodium relictum and anti-Plasmodium elongatum antibodies in sera from experimentally infected ducklings. Whole blood, serum, and dried blood on filter paper gave similar results. The latter was selected for convenience. All birds infected by experimental blood challenge, but not exposed to sporozoites, had detectable antibody (up to 1.0 x 10(-3.8) dilution) reactive with R32tet32, P.F.R27, and CRBCE antigens. Ducklings infected with P. elongatum had higher antibody levels than those infected with P. relictum. In a blind trial, the described ELISA accurately distinguished sera taken from infected and uninfected ducklings. This study provides the first evidence on cross reactivity in the ELISA format between P. falciparum antigens and antibodies induced by P. relictum and P. elongatum in experimentally infected ducklings. The proposed ELISA is fast, easy to perform, reproducible, and requires a minimal amount of equipment. The assay can be used for the detection of P. relictum and P. elongatum antibodies in captive or wild ducks, along with monitoring the level of antibody in selected groups of birds or for surveys of laboratory experiments where evidence of infection is required.

Distribution of repetitive and non-repetitive circumsporozoite protein epitopes on Plasmodium falciparum sporozoites and immunochemical characterization of human malaria antisera

The presence and distribution of circumsporozoite protein (CSP) epitopes located in the repetitive and non-repetitive regions were studied in three Plasmodium falciparum strains, NF54, IFA5 and IFA6. It was found by immunofluorescence, Western blotting and immunoelectron microscopy that mAbs to epitopes of the repetitive domaine bound similarly to the CSP of all three strains. MAbs to epitopes of the flanking regions yielded either some strain differences (mAbs to the C-terminal end), or reacted only in immunofluorescence tests on whole sporozoites (mAbs to the N-terminal end). Human sera from an area endemic for malaria, two of them positive in ELISA on (NANP) 40 and two negative, were tested for their reactivity with epitopes of the flanking regions of the CSP. The presence of antibodies to such epitopes could be demonstrate`d by Western blots and immunocytochemistry independent of the reactivity of the sera to recognize (NANP) 40. All tested bound to salivary gland tissues but not to their secretory product in immunocytochemical experiments.

Strain Specificity in the Liver-Stage Development of Plasmodium Falciparum in Primary Cultures of new World Monkey Hepatocytes

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.

Plasmodium falciparum: administration of anti-sporozoite antibodies during sporogony results in production of sporozoites which are not neutralized by human anti-circumsporozoite protein vaccine sera

Five days after receiving a Plasmodium falciparum NF54 infectious blood meal, Anopheles stephensi mosquitoes were fed rat anti- P. falciparum spprozoite or rabbit anti-R32tet 32 antibodies. Sporozoites isolated from salivary glands were tested by the inhibition of sporozoite invasion (ISI) assay using monoclonal antibody (Mab) 2A10 to P. falciparum circumsporozoite protein or sera from human volunteers immunized with P. falciparum R32tet 32 or (NANP) 3-TT vaccines. Whereas sporozoites from control mosquitoes were neutralized by Mab 2A10 and vaccine sera, only the Mab and not the vaccine sera neutralized sporozoites from immune-fed mosquitoes. The implications of these results in vaccine design and the impact on transmission are discussed.