In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I

In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.     

Delta/notch-like epidermal growth factor (EGF)-related receptor, a novel EGF-like repeat-containing protein targeted to dendrites of developing and adult central nervous system neurons

We have identified a novel epidermal growth factor (EGF)-like repeat-containing single-pass transmembrane protein that is specifically expressed in the developing and mature central nervous system. Sequence analysis revealed that the 10 EGF-like repeats in the extracellular domain are closely related to those of the developmentally important receptor Notch and its ligand Delta. We thus named the molecule Delta/Notch-like EGF-related receptor (DNER). DNER protein is strongly expressed in several types of post-mitotic neurons, including cortical and hippocampal pyramidal neurons, cerebellar granule cells, and Purkinje cells. DNER protein is localized to the dendritic plasma membrane and endosomes and is excluded from the axons, even when overexpressed. The tyrosine-based sorting motif in the cytoplasmic domain is required for dendritic targeting of DNER. Direct in vivo binding of DNER to the coat-associated protein complex AP-1 strongly suggests that DNER undergoes AP-1-dependent sorting to the somatodendritic compartments from the trans-Golgi network and subsequent passage through the endosomal system.