During the bloodstage of malaria infection, the parasite internalizes and degrades massive amounts of hemoglobin from the host red blood cell. Using serial thin-section electron microscopy and threedimensional reconstruction, we demonstrate four independent, but partially overlapping, hemoglobin-uptake processes distinguishable temporally, morphologically, and pharmacologically. Early ring-stage parasites undergo a profound morphological transformation in which they fold, like a cup, onto themselves and in so doing take a large first gulp of host cell cytoplasm. This event, which we term the ''Big Gulp,'' appears to be independent of actin polymerization and marks the first step in biogenesis of the parasite's lysosomal compartment-the food vacuole. A second, previously identified uptake process, uses the cytostome, a well characterized and morphologically distinct structure at the surface of the parasite. This process is more akin to classical endocytosis, giving rise to small (<0.004 fl) vesicles that are marked by the early endosomal regulatory protein Rab5a. A third process, also arising from cytostomes, creates long thin tubes previously termed cytostomal tubes in an actin-dependent manner. The fourth pathway, which we term phagotrophy, is similar to the Big Gulp in that it more closely resembles phagocytosis, except that phagotrophy does not require actin polymerization. Each of these four processes has aspects that are unique to Plasmodium, thus opening avenues to antimalarial therapy.actin ͉ endocytosis ͉ food vacuole ͉ cytosome ͉ transport
Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.
Members of the apicomplexan family of parasites contain morphologically unique secretory organelles termed rhoptries that are essential for host cell invasion. Rhoptries contain internal membranes, and thus resemble multivesicular bodies. To determine whether multivesicular body endosomal intermediates are formed in Apicomplexa, we used the Plasmodium falciparum homolog of the class E gene, Vps4, as a probe. Endogenous P. falciparum Vps4 (PfVps4) localized to the cytoplasm of P. falciparum trophozoites, and transgenic PfVps4 localized to the cytosol in P. falciparum, in the related parasite Toxoplasma gondii and in COS cells. When mutated to block ATP hydrolysis, transiently expressed PfVps4 localized instead to large vesicular structures in P. falciparum. The same construct, and another mutant blocked in ATP binding, generated large cholesterol-enriched multivesicular bodies in both COS cells and T. gondii. Mutant PfVps4 structures in T. gondii co-localized with markers for early endosomes. These results demonstrate a conservation of Vps4 function across wide phylogenetic boundaries, and indicate that endosomal multivesicular bodies form in both P. falciparum and T. gondii.
The development of an alternative source for donor lungs would change the paradigm of lung transplantation. Recent studies have demonstrated the potential feasibility of using decellularized lungs as scaffolds for lung tissue regeneration and subsequent implantation. However, finding a reliable cell source and the ability to scale up for recellularization of the lung scaffold still remain significant challenges. To explore the possibility of regeneration of human lung tissue from stem cells in vitro, populations of lung progenitor cells were generated from human iPSCs. To explore the feasibility of producing engineered lungs from stem cells, we repopulated decellularized human lung and rat lungs with iPSC-derived epithelial progenitor cells. The iPSCs-derived epithelial progenitor cells lined the decellularized human lung and expressed most of the epithelial markers when were cultured in a lung bioreactor system. In decellularized rat lungs, these human-derived cells attach and proliferate in a manner similar to what was observed in the decellularized human lung. Our results suggest that repopulation of lung matrix with iPSC-derived lung epithelial cells may be a viable strategy for human lung regeneration and represents an important early step toward translation of this technology.
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