Cytoadherence and ultrastructure of Plasmodium falciparum-infected erythrocytes from a splenectomized patient.
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In malarial infections of primates, the spleen has been shown to modulate parasite antigen expression on the surfaces of infected erythrocytes. The processes affected include cytoadherence, which is central to the pathophysiology of severe falciparum malaria, and the related phenomenon of rosette formation. In this study, the cytoadherence and rosette formation behaviors of Plasmodium falciparum-infected erythrocytes from a splenectomized patient were examined during the first erythrocytic cycle in vitro. Ultrastructural studies were also performed. Infected erythrocytes were found to cytoadhere to C32 melanoma cells via leukocyte differentiation antigen CD36 but not intercellular adhesion molecule 1. They also displayed on their surfaces electron-dense knobs similar in structure and density to those on infected erythrocytes from intact hosts. These findings may reflect a stable cytoadherent phenotype of the parasite isolate that is unaffected by the absence of the spleen. Alternatively, the modulating role of the spleen may have been assumed by other organs of the mononuclear phagocytic system in a previously infected individual. No rosette formation was observed, but as not all natural isolates form rosettes, this observation may or may not be related to the asplenic status of the patient. Parasite and host factors appear to be important in determining the effect of splenectomy on cytoadherence and rosette formation in human falciparum malaria. (+info)
A role for complement receptor-like molecules in iron acquisition by Candida albicans.
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Candida albicans, an opportunistic fungal pathogen of humans, is dependent upon iron for growth. Consequently, human serum inhibits C. albicans growth due to the presence of high affinity iron-binding proteins that sequester serum iron, making it unavailable for use by the organism. We report that in the inhibitory environment of human serum, the growth of C. albicans can be restored by the addition of exogenous hemoglobin or heme, but not by protoporphyrin IX, the heme precursor that does not contain iron. We further report that C. albicans can utilize cell surface proteins that are homologues of the mammalian complement receptors (CR) to rosette complement-coated red blood cells (RBC) and obtain RBC-derived iron for growth. The ability of Candida to acquire RBC-derived iron under these conditions is dependent upon Candida-RBC rosetting mediated by CR-like molecules. Unopsonized RBC do not support Candida growth in serum, and restoration of Candida growth in serum by complement-opsonized RBC is inhibited by monoclonal antibodies to the human CR type 3 (CR3). In addition, activation of the human alternative pathway of complement by Candida leads to "bystander" deposition of C3 fragments on the surface of autologous, unopsonized RBC, generating the ligands necessary for Candida-RBC rosetting. These results suggest that C. albicans has evolved a unique strategy for acquiring iron from the host, which exploits the host complement system, and which may contribute to the pathogenic potential of the organism. (+info)
Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum.
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Erythrocyte binding antigen 175 (EBA-175) is a P. falciparum protein that binds the major glycoprotein found on human erythrocytes, glycophorin A, during invasion. Here we present the crystal structure of the erythrocyte binding domain of EBA-175, RII, which has been established as a vaccine candidate. Binding sites for the heavily sialylated receptor glycophorin A are proposed based on a complex of RII with a glycan that contains the essential components required for binding. The dimeric organization of RII displays two prominent channels that contain four of the six observed glycan binding sites. Each monomer consists of two Duffy binding-like (DBL) domains (F1 and F2). F2 more prominently lines the channels and makes the majority of the glycan contacts, underscoring its role in cytoadherence and in antigenic variation in malaria. Our studies provide insight into the mechanism of erythrocyte invasion by the malaria parasite and aid in rational drug design and vaccines. (+info)
Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions.
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Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX1, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-1, a neural RNA-binding protein, but few cells expressed further differentiation markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin, alpha fetoprotein, or the pluripotent marker Oct4. Further differentiation of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number. (+info)
Clinical and molecular aspects of severe malaria.
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The erythrocytic cycle of Plasmodium falciparum presents a particularity in relation to other Plasmodium species that infect man. Mature trophozoites and schizonts are sequestered from the peripheral circulation due to adhesion of infected erythrocytes to host endothelial cells. Modifications in the surface of infected erythrocytes, termed knobs, seem to facilitate adhesion to endothelium and other erythrocytes. Adhesion provides better maturation in the microaerophilic venous atmosphere and allows the parasite to escape clearance by the spleen which recognizes the erythrocytes loss of deformability. Adhesion to the endothelium, or cytoadherence, has an important role in the pathogenicity of the disease, causing occlusion of small vessels and contributing to failure of many organs. Cytoadherence can also describe adhesion of infected erythrocytes to uninfected erythrocytes, a phenomenon widely known as rosetting. Clinical aspects of severe malaria, as well as the host receptors and parasite ligands involved in cytoadherence and rosetting, are reviewed here. The erythrocyte membrane protein 1 of P. falciparum (PfEMP1) appears to be the principal adhesive ligand of infected erythrocytes and will be discussed in more detail. Understanding the role of host receptors and parasite ligands in the development of different clinical syndromes is urgently needed to identify vaccination targets in order to decrease the mortality rates of this disease. (+info)
Rapid switching to multiple antigenic and adhesive phenotypes in malaria.
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Adhesion of parasitized erythrocytes to post-capillary venular endothelium or uninfected red cells is strongly implicated in the pathogenesis of severe Plasmodium falciparum malaria. Neoantigens at the infected red-cell surface adhere to a variety of host receptors, demonstrate serological diversity in field isolates and may also be a target of the host-protective immune response. Here we use sequential cloning of P. falciparum by micromanipulation to investigate the ability of a parasite to switch antigenic and cytoadherence phenotypes. Our data show that antigens at the parasitized cell surface undergo clonal variation in vitro in the absence of immune pressure at the rate of 2% per generation with concomitant modulations of the adhesive phenotype. A clone has the potential to switch at high frequency to a variety of antigenic and adhesive phenotypes, including a new type of cytoadherence behaviour, 'auto-agglutination' of infected erythrocytes. This rapid appearance of antigenic and functional heterogeneity has important implications for pathogenesis and acquired immunity. (+info)
Follicular dendritic-like cells derived from human monocytes.
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BACKGROUND: Follicular dendritic cells (FDCs) play a central role in controlling B-cell response maturation, isotype switching and the maintenance of B-cell memory. These functions are based on prolonged preservation of antigen and its presentation in its native form by FDCs. However, when entrapping entire pathogens, FDCs can turn into dangerous long-term reservoirs that may preserve viruses or prions in highly infectious form. Despite various efforts, the ontogeny of FDCs has remained elusive. They have been proposed to derive either from bone marrow stromal cells, myeloid cells or local mesenchymal precursors. Still, differentiating FDCs from their precursors in vitro may allow addressing many unsolved issues associated with the (patho-) biology of these important antigen-presenting cells. The aim of our study was to demonstrate that FDC-like cells can be deduced from monocytes, and to develop a protocol in order to quantitatively generate them in vitro. RESULTS: Employing highly purified human monocytes as a starter population, low concentrations of Il-4 (25 U/ml) and GM-CSF (3 U/ml) in combination with Dexamethasone (Dex) (0.5 microM) in serum-free medium trigger the differentiation into FDC-like cells. After transient de-novo membrane expression of alkaline phosphatase (AP), such cells highly up-regulate surface expression of complement receptor I (CD35). Co-expression of CD68 confirms the monocytic origin of both, APpos and CD35pos cells. The common leukocyte antigen CD45 is strongly down-regulated. Successive stimulation with TNF-alpha up-regulates adhesion molecules ICAM-1 (CD54) and VCAM (CD106). Importantly, both, APpos as well as APneg FDC-like cells, heterotypically cluster with and emperipolese B cells and exhibit the FDC characteristic ability to entrap functionally preserved antigen for prolonged times. Identical characteristics are found in monocytes which were highly expanded in vitro by higher doses of GM-CSF (25 U/ml) in the absence of Dex and Il-4 before employing the above differentiation cocktail. CONCLUSION: In this work we provide evidence that FDC-like cells can be derived from monocytes in vitro. Monocyte-derived FDC-like cells quantitatively produced offer a broad utility covering basic research as well as clinical application. (+info)
Plasmodium falciparum: Rosettes do not protect merozoites from invasion-inhibitory antibodies.
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Rosetting is a parasite adhesion phenotype associated with severe malaria in African children. Why parasites form rosettes is unknown, although enhanced invasion or immune evasion have been suggested as possible functions. Previous work showed that rosetting does not enhance parasite invasion under standard in vitro conditions. We hypothesised that rosetting might promote invasion in the presence of host invasion-inhibitory antibodies, by allowing merozoites direct entry into the erythrocytes in the rosette and so minimising exposure to plasma antibodies. We therefore investigated whether rosetting influences invasion in the presence of invasion-inhibitory antibodies to MSP-1. We found no difference in invasion rates between isogenic rosetting and non-rosetting lines from two parasite strains, R29 and TM284, in the presence of MSP-1 antibodies (P = 0.62 and P = 0.63, Student's t test, TM284 and R29, respectively). These results do not support the hypothesis that rosettes protect merozoites from inhibitory antibodies during invasion. The biological function of rosetting remains unknown. (+info)