Ajoene (4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a product initially isolated from extracts of garlic (Alium sativum), was tested for its antimalarial activity in vivo in a well-characterized murine model. A single ajoene dose of 50 mg/kg, on the day of infection, suppressed the development of parasitemia; there were no obvious acute toxic effects from the tested dose. The combination of ajoene (50 mg/kg) and chloroquine (4.5 mg/kg), given as a single dose on the day of the infection, completely prevented the subsequent development of parasitemia in treated mice.Malaria parasites resistant to various drugs are widespread in South America, Asia, and Africa (15). In particular, the chloroquine (CQ)-resistant strains of Plasmodium falciparum have become a major public health problem around the world (11,15). Hence, the search for new antimalarial therapies is a high-priority task for the control of the disease. Ajoene (4,5,9-trithiadodeca-1,6,11-triene 9-oxide), an organosulfur compound derived from garlic (Allium sativum) (1, 7), is a well-known inhibitor of platelet activation (2-4) and also has significant antifungal (10, 12, 16), antitrypanosomal (14), and antiviral (13) activities.We investigated the potential anti-Plasmodium berghei activity of ajoene in vivo. The results show that when used alone, ajoene displays a moderate anti-P. berghei activity. However, when used in combination with a noneffective dose of CQ, ajoene synergistically enhances the susceptibility of the parasite to this drug. MATERUILS AND METHODSAjoene. Synthetic ajoene (Mr, 234) was prepared as previously described (1, la, 3), dissolved in ethanol, and diluted eightfold with Intralipid (200 g of fractionated soybean, 12 g of fractionated egg phospholipids, 22 g of glycerol USP, 1,000 ml of water q.s.p) (Kavi Vitrum, Stockholm, Sweden). The final concentration of ethanol was less than 1% (vol/vol Animal experiments. All experimental mice were infected at random before being divided into groups of 7 to 10. Mice were treated by intraperitoneal injection of ajoene given on the day of inoculation (day 0). The time between the inoculation of parasites and drug injection was 60 min. The effectiveness of CQ alone at a dose of 4.5 mg/kg (8) or in combination with various concentrations of ajoene was studied by using the same experimental protocol described for ajoene. CQ diphosphate (CQ; Sigma Chemical Co.) was diluted in 0.9% (wt/vol) NaCl to give the dose required in 0.1 ml for 10 g of mouse and was administered intraperitoneally. Experiments were repeated at least three times. Data from a representative experiment are given. RESULTSDrug toxicity. On the basis of the results of a set of preliminary experiments, ajoene doses of up to 50 mg/kg were used. No signs of toxicity were observed in 30 days.Inhibition of parasite growth by ajoene. In BALB/c mice infected with 105 parasitized erythrocytes, systemic parasitemia became apparent (1%) on day 2 and increased to 24 ± 2.8% infected cells on day 6 after the inoculation. Ajoene (50 mg/kg) given ...

Blood stages of Plasmodium vivax induce the development of caveolae and caveola-vesicle complexes (CVC) in the membrane of their host erythrocyte. Caveolae are found in almost all types of cells and are involved in endogenous processes as calcium and cholesterol homeostasis, cell signalling, transporting, ligand internalization and transcytosis of serum components. Major structural components of caveolae are the proteins caveolins and flotillins. The functional role of caveolae in the P. vivax-infected erythrocyte is not properly understood. As these organelles have been shown to contain malaria antigens, it has been suggested that they are involved in the transport and release of specific parasite antigens from the infected erythrocyte and in the uptake of plasma proteins. Using specific antibodies to classical caveolae proteins and an immunolocalization approach, we found caveolin-2, caveolin-3, and flotillin-2 in the vesicle profiles and some CVC of P. vivax-infected erythrocytes. Caveolin-1-3 were not found in uninfected erythrocytes. This is the first report of identification and localization of caveolins in the CVC present in erythrocytes infected with P. vivax, thereby providing evidence of the role of this particular organelle in the protein-trafficking pathway that connect parasite-encoded proteins with the erythrocyte cytoplasm and the cell surface throughout the asexual blood cycle of vivax malaria parasite.

Protein deprived C57BL/6 mice infected with 10(3) amastigotes of Leishmania mexicana showed early arrest of lesion progression during the first 5 weeks of infection with subsequent development of progressive non-healing lesions. In contrast, well nourished mice similarly infected developed gradual healing lesions. The early resistance of malnourished mice to 10(3) amastigotes was overcome by a larger dose. After a primary inoculation with 10(3) amastigotes protein deprived mice failed to express protective immunity to a challenge inoculum given at 5 weeks of infection. When challenge was delayed until 10 weeks, protein deprived mice developed lesions at the site of challenge which tended to regress but were unable to manifest the high level of protective immunity seen in normally nourished reinfected controls. A challenge infection given at 5 or 10 weeks prejudiced the control of primary lesions particularly in the group of protein deprived mice challenged at 10 weeks. Equivalent levels of specific delayed hypersensitivity responses were found in protein deprived and normally nourished uninfected mice immunized with killed parasites which imply that the impaired protective immunity observed in protein deprived mice is not due to a deleterious effect of protein deprivation on the ability of the host to develop cellular responses such as delayed type hypersensitivity to Leishmania antigens.

We investigated the secretory traffic of a Plasmodium vivax antigen (Pv-148) synthesised by the parasite during the blood cycle, exported into the host cell cytosol and then transported to the surface membrane of the infected erythrocyte. Studies of the ultrastructure of erythrocytes infected with P. vivax showed that intracellular schizogony is accompanied by the generation of parasite-induced membrane profiles in the erythrocyte cytoplasm. These structures are detectable soon after the parasite invades the erythrocyte and develop an elaborate organisation, leading to a tubovesicular membrane (TVM) network, in erythrocytes infected with mature trophozoites. Interestingly, the clefts formed stacked, flattened cisternae resembling a classical Golgi apparatus. The TVM network stained with the fluorescent Golgi marker Bodipy-ceramide. Specific immunolabelling showed that Pv-148 was transferred from the parasite to the erythrocyte surface membrane via the clefts and the TVM network. These findings suggest that the TVM network is part of the secretory pathways involved in parasite protein transport across the Plasmodium-infected erythrocyte and that Pv- 148 may represent a marker that links the parasite with the host cell cytoplasm and, in turn, with the extracellular milieu.

A monoclonal antibody raised by immunization of BALB/c mice with erythrocytic stages of Plasmodium vivax was shown to react with asexual erythrocytic stages of P. chabaudi. The cross-reactivity molecules are antigens of 200 and 148 kDa in P. vivax and of 190 and 70 kDa in P. chabaudi. Immunofluorescence studies of the erythrocytic stages of P. vivax and P. chabaudi indicated that expression of these antigens increased as the parasites' developed from the ring stage to the schizont stage. In the mature trophozoites of P. chabaudi, immunoelectron microscopy revealed clusters of antigen distributed in the cytoplasm of the parasitized erythrocyte. In the schizont, packets of antigen were found associated with the parasitophorous vacuole and the cytoplasm of the infected host cell.