Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.

The potential benefits to health of antioxidant enzymes supplied either through dietary intake or supplementation is still a matter of controversy. The development of dietary delivery systems using wheat gliadin biopolymers as a natural carrier represents a new alternative. Combination of antioxidant enzymes with this natural carrier not only delayed their degradation (i.e. the superoxide dismutase, SOD) during the gastrointestinal digestive process, but also promoted, in vivo, the cellular defences by strengthening the antioxidant status. The effects of supplementation for 28 days with a standardized melon SOD extract either combined (Glisodin) or not with gliadin, were evaluated on various oxidative-stress biomarkers. As already described there was no change either in superoxide dismutase, catalase or glutathione peroxidase activities in blood circulation or in the liver following non-protected SOD supplementation. However, animals supplemented with Glisodin showed a significant elevation in circulated antioxidant enzymes activities, correlated with an increased resistance of red blood cells to oxidative stress-induced hemolysis. In the presence of Sin-1, a chemical donor of peroxynitrites, mitochondria from hepatocytes regularly underwent membrane depolarization as the primary biological event of the apoptosis cascade. Hepatocytes isolated from animals supplemented with Glisodin presented a delayed depolarization response and an enhanced resistance to oxidative stress-induced apoptosis. It is concluded that supplementation with gliadin-combined standardized melon SOD extract (Glisodin) promoted the cellular antioxidant status and protected against oxidative stress-induced cell death.

The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. The steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo. INTRODUCTIONThe notion that Anopheles mosquitoes inject Plasmodium sporozoites into the dermis of the host, rather than directly into the blood circulation, was first suggested in the 1930s 1 and has since received experimental confirmation 2-4 . Sporozoites are ejected when the mosquito salivates 5 , especially when it probes the dermis, searching for a blood source. However, given the small number of sporozoites injected through its bite and the highly motile behavior of these sporozoites, the dermal phase of sporozoite infection is still poorly characterized. Only now, with the development of tools for studying the fast dynamics of sporozoites in real time, can their exact in vivo fate be analyzed.A fundamental feature of the malarial sporozoite is its motility and migratory behavior. Like other apicomplexan parasites, this needle-shaped cell (10 mm in length, 1 mm in width) moves by gliding over a substrate at very high speeds (up to 4 mm s À1 ). The sporozoite and its gliding properties have mainly been studied in species of Plasmodium that infect rodents (P. berghei and P. yoelii), which offer, over the human-infecting parasite species, the advantages of safety in handling the parasites and ease in manipulating their genome. The rodent systems offer an additional key advantage, the possibility of studying parasites in as near as possible to their natural environment. A number of wild-type fluorescent sporozoites expressing a fluorescent marker (GFP or RFP) through a variety of stage-specific or constitutive promoters are now available in both P. berghei 6,7 and P. yoelii 8 , which can now be imaged in the dermis or liver of rodent hosts 9-12 .Imaging techniques have been revolutionized by advancements in both microscope instrumentation and data collection/processing software. Although one-photon methods are more limited than two-phot...

The form of the malaria parasite inoculated by the mosquito, called the sporozoite, transforms inside the host liver into thousands of a new form of the parasite, called the merozoite, which infects erythrocytes. We present here a protocol to visualize in vivo the behavior of Plasmodium berghei parasites in the hepatic tissue of the murine host. The use of GFP-expressing parasites and a high-speed spinning disk confocal microscope allows for the acquisition of four-dimensional images, which provide a time lapse view of parasite displacement and development in tissue volumes. These data can be analyzed to give information on the early events of sporozoite penetration of the hepatic tissue, that is, sporozoite gliding in the liver sinusoids, crossing the sinusoidal barrier, gliding in the parenchyma and traversal of hepatocytes, and invasion of a final hepatocyte, as well as the terminal events of merosome and merozoite release from infected hepatocytes. Combined with the use of mice expressing fluorescent cell types or cell markers, the system will provide useful information not only on the primary infection process, but also on parasite interactions with the host immune cells in the liver.