Potential role of Plasmodium falciparum exported protein 1 in the chloroquine mode of action.

In the human malaria parasite Plasmodium falciparum, membrane glutathione S-transferases (GST) have recently emerged as potential cellular detoxifying units and as drug target candidates with the artemisinin (ART) class of antimalarials inhibiting their activity at single-digit nanomolar potency when activated by iron sources such as cytotoxic hematin. Here we put forward the hypothesis that the membrane GST Plasmodium falciparum exported protein 1 (PfEXP1, PF3D7_1121600) might be directly involved in the mode of action of the unrelated antimalarial 4-aminoquinoline drug chloroquine (CQ). Along this line we report potent biochemical inhibition of membrane glutathione S-transferase activity in recombinant PfEXP1 through CQ at half maximal inhibitory CQ concentrations of 9.02 nM and 19.33 nM when using hematin and the iron deficient 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, respectively. Thus, in contrast to ART, CQ may not require activation through an iron source such as hematin for a potent inhibition of membrane GST activity. Arguably, these data represent the first instance of low nanomolar inhibition of an essential Plasmodium falciparum enzyme through a 4-aminoquinoline and might encourage further investigation of PfEXP1 as a potential CQ target candidate.

Potential therapeutic use of herbal extracts in trypanosomiasis.

The aim of the present study was to evaluate the effects of crude extracts from Handroanthus impetiginosa, Ageratum conyzoides, and Ruta graveolens on Leishmania amazonensis and Trypanosoma cruzi infection in vitro. The results showed that the extracts caused significant toxicity in promastigotes and trypomastigotes. A significant decrease in the rate of cell invasion by pretreated trypomastigotes and promastigotes was also observed. The extracts caused a significant reduction of the multiplication of intracellular amastigotes of both parasites. Therefore, these herbal extracts may be potential candidates for the development of drugs for the treatment of leishmaniasis and Chagas disease.

A thrombospondin structural repeat containing rhoptry protein from Plasmodium falciparum mediates erythrocyte invasion.

Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin-treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy for the development of vaccines against blood stage malaria parasites.

EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2.

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils in solution. As this propensity of MSP2 to form fibrils in solution has the potential to impede its development as a vaccine candidate, finding an inhibitor that inhibits fibrillogenesis may enhance vaccine development. We have shown previously that EGCG inhibits the formation of MSP2 fibrils. Here we show that EGCG can alter the ß-sheet-like structure of the fibril and disaggregate pre-formed fibrils of MSP2 into soluble oligomers. The fibril remodelling effects of EGCG and other flavonoids were characterised using Thioflavin T fluorescence assays, electron microscopy and other biophysical methods.

Plasmodium vivax DBP binding to Aotus nancymaae erythrocytes is Duffy antigen dependent.

Plasmodium vivax is the second leading cause of malaria worldwide. Invasion of human erythrocytes by P. vivax merozoites is dependent upon the interaction between the parasite Duffy binding protein (PvDBP) and the erythrocyte Duffy antigen receptor. Therefore, disruption of this vital interaction is an attractive target for therapeutic intervention. Although Aotus nancymaae is a commonly used primate model for human P. vivax infections, it has not been confirmed that the interaction between Ao. nancymaae erythrocytes and P. vivax is Duffy antigen dependent. Our results indicate that normal Ao. nancymaae erythrocytes readily bind to PvDBPII and that this binding is completely abolished with chymotrypsin treatment of the erythrocytes. Furthermore, the results of our inhibition assays show a dose-dependent decrease in binding with increasing amounts of anti-PvDBPII polyclonal rabbit sera or anti-Fy6 monoclonal antibody. These data indicate that the interaction between Ao. nancymaae erythrocytes and P. vivax DBPII is Duffy antigen dependent, validating this model system for in vivo studies of anti-PvDBP inhibition.

Trypanosoma cruzi: parasite antigens sequestered in heart interstitial dendritic cells are related to persisting myocarditis in benznidazole-treated mice.

We investigated whether sequestered Trypanosoma cruzi antigens found in heart interstitial dendritic cells (IDCs) contribute to the residual myocarditis found in mice following treatment with benznidazole, a specific chemotherapeutic drug. IDCs are antigen-presenting cells that are MHC-II-receptor dependent. Swiss mice were divided into two experimental groups: the 1st group was infected with the Colombian strain of T. cruzi, which is resistant to treatment with benznidazole, and the 2nd group was infected with clone 21SF-C 3, which has a medium susceptibility to the drug. Treatment of the Colombian strain group started on the 120th day post-infection and for the 21SF-C3 strain group treatment was started on the 90th day. In both groups, treatment lasted for 90 days. The animals were sacrificed either 150 or 200 days post-treatment. The myocardium was analysed by immunohistochemistry using anti-MAC3, 33D1, CD11b and CD11c monoclonal antibodies for IDCs or anti-T. cruzi purified antibodies. Parasite antigens were expressed on the IDC membranes in both treated and untreated mice. Myocarditis subsided following treatment, evidenced by both histological and morphometrical evaluation. A reduction in the number of IDCs carrying T. cruzi antigens in the treated group indicates that the elimination of parasites influences antigen presentation with concomitant decreases in inflammation. There is a correlation between the presence of T. cruzi antigens in these cells and the chronic focal, residual myocarditis seen in treated mice.

Trypanosoma cruzi: parasite antigens sequestered in heart interstitial dendritic cells are related to persisting myocarditis in benznidazole-treated mice

We investigated whether sequestered Trypanosoma cruzi antigens found in heart interstitial dendritic cells (IDCs) contribute to the residual myocarditis found in mice following treatment with benznidazole, a specific chemotherapeutic drug. IDCs are antigen-presenting cells that are MHC-II-receptor dependent. Swiss mice were divided into two experimental groups: the 1st group was infected with the Colombian strain of T. cruzi, which is resistant to treatment with benznidazole, and the 2nd group was infected with clone 21SF-C 3, which has a medium susceptibility to the drug. Treatment of the Colombian strain group started on the 120th day post-infection and for the 21SF-C3 strain group treatment was started on the 90th day. In both groups, treatment lasted for 90 days. The animals were sacrificed either 150 or 200 days post-treatment. The myocardium was analysed by immunohistochemistry using anti-MAC3, 33D1, CD11b and CD11c monoclonal antibodies for IDCs or anti-T. cruzi purified antibodies. Parasite antigens were expressed on the IDC membranes in both treated and untreated mice. Myocarditis subsided following treatment, evidenced by both histological and morphometrical evaluation. A reduction in the number of IDCs carrying T. cruzi antigens in the treated group indicates that the elimination of parasites influences antigen presentation with concomitant decreases in inflammation. There is a correlation between the presence of T. cruzi antigens in these cells and the chronic focal, residual myocarditis seen in treated mice.

Blood groups and malaria: fresh insights into pathogenesis and identification of targets for intervention.

PURPOSE OF REVIEW: This review summarizes recent advances in our understanding of the interaction between malaria parasites and blood group antigens and discusses how the knowledge gleaned can be used to target the development of new antimalarial treatments and vaccines. RECENT FINDINGS: Studies of the interaction between Plasmodium vivax and the Duffy antigen provide the clearest example of the potential for basic research on blood groups and malaria to be translated into a vaccine that could have a major impact on global health. Progress is also being made in understanding the effects of other blood group antigens on malaria. After years of controversy, the effect of ABO blood groups on falciparum malaria has been clarified, with the non-O blood groups emerging as significant risk factors for life-threatening malaria, through the mechanism of enhanced rosette formation. The Knops blood group system may also influence malaria susceptibility, although conflicting results from different countries mean that further research is required. Unanswered questions remain about the interactions between malaria parasites and other blood group antigens, including the Gerbich, MNS and Rhesus systems. SUMMARY: The interplay between malaria parasites and blood group antigens remains a fascinating subject with potential to contribute to the development of new interventions to reduce the global burden of malaria.

Therapeutic efficacy of artemether-lumefantrine in uncomplicated falciparum malaria in India.

BACKGROUND: Artemisinin-based combination therapy (ACT) is the treatment of choice for uncomplicated falciparum malaria. Artemether-lumefantrine (AL), a fixed dose co-formulation, has recently been approved for marketing in India, although it is not included in the National Drug Policy for treatment of malaria. Efficacy of short course regimen (4 x 4 tablets of 20 mg artemether plus 120 mg lumefantrine over 48 h) was demonstrated in India in the year 2000. However, low cure rates in Thailand and better plasma lumefantrine concentration profile with a six-dose regimen over three days, led to the recommendation of higher dose globally. This is the first report on the therapeutic efficacy of the six-dose regimen of AL in Indian uncomplicated falciparum malaria patients. The data generated will help in keeping the alternative ACT ready for use in the National Programme as and when required. METHODS: One hundred and twenty four subjects between two and fifty-five years of age living in two highly endemic areas of the country (Assam and Orissa) were enrolled for single arm, open label prospective study. The standard six-dose regimen of AL was administered over three days and was followed-up with clinical and parasitological evaluations over 28 days. Molecular markers msp-1 and msp-2 were used to differentiate the recrudescence and reinfection among the study subjects. In addition, polymorphism in pfmdr1 was also carried out in the samples obtained from patients before and after the treatment. RESULTS: The PCR corrected cure rates were high at both the sites viz. 100% (n = 53) in Assam and 98.6% (n = 71) in Orissa. The only treatment failure case on D7 was a malnourished child. The drug was well tolerated with no adverse events. Patients had pre-treatment carriage of wild type codons at positions 86 (41.7%, n = 91) and 184 (91.3%, n = 91) of pfmdr1 gene. CONCLUSION: AL is safe and effective drug for the treatment of acute uncomplicated falciparum malaria in India. The polymorphism in pfmdr1 gene is not co-related with clinical outcome. However, treatment failure can also occur due to incomplete absorption of the drug as is suspected in one case of failure at D7 in the study. AL can be a viable alternative of artesunate plus sulphadoxine/pyrimethamine (AS + SP), however, the drug should be used rationally and efficacy needs to be monitored periodically.

Enzymatic inhibitory activity and trypanocidal effects of extracts and compounds from Siphoneugena densiflora O. Berg and Vitex polygama Cham.

Hexanic, methanolic, and hydroalcoholic extracts, and 34 isolated compounds from Vitex polygama Cham. (Lamiaceae, formely Verbenaceae) and Siphoneugena densiflora O. Berg (Myrtaceae) were screened for their trypanocidal effects on bloodstream forms of Trypanosoma cruzi and T brucei, as well as for their enzymatic inhibitory activities on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and trypanothione reductase (TR) enzymes from T cruzi and adeninephosphoribosyl transferase (APRT) enzyme from Leishmania tarentolae. In general, polar extracts displayed strong effects and some of the tested compounds have shown good results in comparison to positive controls of the bioassays.

Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum.

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.

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.

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.

Antibodies from malaria-exposed pregnant women recognize trypsin resistant epitopes on the surface of Plasmodium falciparum-infected erythrocytes selected for adhesion to chondroitin sulphate A.

BACKGROUND: The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM). METHODS: Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for adhesion to CSA. The effect of proteolytic digestion of parasite erythrocyte surface antigens on surface IgG binding and adhesion to CSA and hyaluronic acid (HA) was also studied. RESULTS: P. falciparum infected erythrocytes selected for adhesion to CSA were found to express trypsin-resistant VSA that are the target of naturally acquired antibodies from pregnant women living in a malaria endemic region of Ghana. However in vitro adhesion to CSA and HA was relatively trypsin sensitive. An improved labelling technique for the detection of VSA expressed by CSA binding isolates has also been described. CONCLUSION: The VSA expressed by CSA binding P. falciparum isolates are currently considered potential targets for a vaccine against PAM. This study identifies discordance between the trypsin sensitivity of CSA binding and surface recognition of CSA selected parasites by serum IgG from malaria exposed pregnant women. Thus, the complete molecular definition of an antigenic P. falciparum erythrocyte surface protein that can be used as a malaria in pregnancy vaccine has not yet been achieved.

A role for tertiary structure in the generation of antigenic diversity and molecular association of the Tams1 polypeptide in Theileria annulata.

The major merozoite-piroplasm surface antigen (mMPSA) of Theileria annulata, Tams1, is known to be antigenically diverse. The possession of variable N-linked glycosylation sites and removal of monoclonal antibody 5E1 reactivity by mild periodate treatment suggested, previously, that divergent epitopes may be conferred by secondary modification. This study has shown that monoclonal antibody 5E1 and polyspecific antisera raised against the native protein react against divergent amino acid epitopes that are dependent on a molecular conformation that is sensitive to periodate. Therefore, no experimental evidence exists to confirm the sequence prediction that Tams1 undergoes N-linked glycosylation. Data is also presented indicating that the conformation of the antigen results in presentation of divergent regions on the external surface of the molecule, while conserved regions are more likely to be internal and hidden. In addition, non-reducing SDS-PAGE analysis demonstrated that Tams1 can undergo molecular association to form homo-dimers, trimers and multimers. The potential influence of tertiary structure and inter-molecular association on Tams1 diversity and function is discussed.

Structural and biochemical alterations in Giardia lamblia cysts exposed to ozone.

Because of its efficacy in inactivating waterborne protozoan cysts and oocysts, ozone is frequently used for disinfection of drinking water. The effect of ozone on cysts of Giardia lamblia was investigated in gerbils (Meriones unguiculatus), using an infectivity assay by scanning electron microscopy, immunoblotting, and flow cytometry. Cysts recovered from experimentally infected gerbils were exposed to an initial ozone concentration of 1.5 mg/L for 0, 30, 60, and 120 sec. This treatment resulted in a dose-dependent reduction in cysts concentration, loss of infectivity in gerbils, and profound structural modifications to the cyst wall. Exposure for 60 sec or longer resulted in extensive protein degradation and in the disappearance of a cyst wall and a trophozoite antigen.

Enhanced immunostimulant activity and protective effect of a synthetic lipopeptide after liposomization against Plasmodium berghei infection in mice.

The immunostimulant activity of non-pyrogenic, sugar-free immunomodulator lipopeptide, Ala-D-Glu(Gly-Lys-CO.C11H23)-NH2 (comp. no 84/201), and its liposomized formulation has been studied. Liposomization of this lipopeptide significantly enhanced its antigen specific as well as nonspecific immune responses, as compared to the free lipopeptide. The liposomized formulation of lipopeptide significantly stimulated both the antibody and delayed-type hypersensitivity responses in Balb/c mice, and also enhanced nonspecifically the macrophage migration index, phagocytic activity and incorporation of 14C glucosamine in peritoneal macrophages of the mice that received pretreatment with this preparation. Further, the mice that received pretreatment with the liposomized preparation strongly resisted lethal P. berghei infection and consequently survived for longer period of times. These results indicate that liposomization of the compound no 84/201 significantly improves its ability to enhance not only antigen-specific immune response but also the nonspecific host's resistance against infections.

Immunological selection for Leishmania (Viannia) braziliensis antigens.

Comparative ELISA and selective immunoblotting procedures were used in attempts to identify differential serological indicators of infection with the Leishmania (Viannia) braziliensis complex, infection with the L. braziliensis species, and therapeutic cure of localized or mucocutaneous leishmaniasis (LCL or MCL). Although mean ELISA absorbance values were significantly higher for MCL sera than for LCL sera, absorbance could not be used as a reliable indicator of the clinical form of disease. Immunoblotting profiles were similar with sera from MCL and LCL. Pre-adsorption with heterologous trypanosomatid antigens indicated that recognition of antigens of about 56, 60, 66, 72, 88 and 110 kDa might be specific to the subgenus Viannia. In two-colour, sequential, dual ELISA-based immunoblotting, no antigens recognized only by sera from MCL patients were detected. After glucantime therapy, immunoblotting profiles with LCL sera were reduced both in intensity and in the range of antigens detected; a 104-kDa antigen was newly detected with post-treatment LCL sera. Overall, the results show the value of differential immunological detection strategies and support the close relationship between species of the subgenus Viannia but fail to indicate a prognostic antigen for MCL.

Structure of the glycosylphosphatidylinositol-anchor of the trans-sialidase from Trypanosoma cruzi metacyclic trypomastigote forms.

Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi shed a glycoprotein, the shed acute phase antigen, that is responsible for the trans-sialidase activity. In the present work the structure of the glycosylphosphatidylinositol membrane anchor of the trans-sialidase isolated from metacyclic forms was determined. Parasites were metabolically labelled with [9, 10(n)3H]-palmitic acid and the glycoprotein was purified by immunoprecipitation with a monoclonal antibody directed against the repetitive aminoacid sequence. Treatment of the glycoprotein with phosphatidylinositol phospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that comigrated in TLC with a standard of ceramide. No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase isolated from culture-derived trypomastigotes where both lipids were found. Chemical and chromatographic analysis showed that the lipid moiety is palmitoyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine. The glycan constituent of the glycosylphosphatidylinositol-anchor was analysed by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid. A major oligosaccharide was obtained and enzymatic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substituted by an alpha-galactose. A comparison between the lipid constituent of the glycosylphosphatidylinositol anchor of several proteins and their spontaneous shedding by the action of an endogenous PI-PLC was made.