Summary Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation and autophagy. Notably, the pro-death protein p53 was substantially decreased in infected hepatocytes, suggesting it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver stage parasite burden whereas p53 knockout mice suffered increased liver stage burden. Furthermore, boosting p53 levels using the small molecule Nutlin-3 dramatically reduced liver stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis.

BackgroundMalaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes.MethodsHere is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants.ResultsThis method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates.ConclusionsThis novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.

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Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.

SUMMARY1. Cell pairs were isolated from ventricles ofadult rat hearts so as to study cell-to-cell coupling.2. Both cells of each pair were impaled with micro-electrodes connected to balanced bridge circuits. Rectangular current pulses were passed and the resulting voltage deflexions monitored.3. The data were analysed in terms of a delta configuration of three resistive elements, the resistances of the non-junctional membrane of cell 1 and cell 2 (rmi and rm, 2)' and the resistance of the nexal membrane (rn).4. The nexal membrane resistance was found to be insensitive to voltage gradients across the non-junctional membrane (range examined: -70 to -10 mV) and direction of current flow.

APAF1, encoding the protein apoptosis protease activating factor 1 (Apaf-1), has recently been established as a chromosome 12 gene conferring predisposition to major depression in humans. The molecular phenotypes of Apaf-1 variants were determined by in vitro reconstruction of the apoptosome complex in which Apaf-1 activates caspase 9 and thus initiates a cascade of proteolytic events leading to apoptotic destruction of the cell. Cellular phenotypes were measured using a yeast heterologous expression assay in which human Apaf-1 and other proteins necessary to constitute a functional apoptotic pathway were overexpressed. Apaf-1 variants encoded by APAF1 alleles that segregate with major depression in families linked to chromosome 12 shared a common gain-of-function phenotype in both assay systems. In contrast, other Apaf-1 variants showed neutral or loss-of-function phenotypes. The depression-associated alleles thus have a common phenotype that is distinct from that of non-associated variants. This result suggests an etiologic role for enhanced apoptosis in major depression.

Candida albicans is a commensal and a common constituent of the human microbiota; however, it can become pathogenic and cause infections in both immunocompetent and immunocompromised people. C. albicans exhibits remarkable metabolic versatility as it can colonize multiple body sites as a commensal or pathogen. Understanding how C. albicans adapts metabolically to each ecological niche is essential for developing novel therapeutic approaches. Purine metabolism has been targeted pharmaceutically in several diseases; however, the regulation of this pathway has not been fully elucidated in C. albicans. Here, we report how C. albicans controls the AMP de novo biosynthesis pathway in response to purine availability. We show that the lack of the transcription factors Grf10 and Bas1 leads to purine metabolic dysfunction, and this dysfunction affects the ability of C. albicans to establish infections.

It seems that LC has a lower rate of adhesion formation than the conventional open technique. Complications such as bleeding or laceration of the liver bed during LC can enhance adhesion formation. No adhesion formation can be mentioned in relation to gallbladder perforation during LC.