The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy . As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca" concentration and fails if this ion is replaced by Mg`or Ba ++ . The removal of Ca" and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance . Resealing was achieved within 10-15 min by restoring Ca" . Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca", was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components . Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction . The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca" on the extracellular side and low Ca'c oncentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7 .5°C) . Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components . The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels ; the Na'/Cl -and the cation/cation selectivity were recovered with the same time-course as the electrical resistance . The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article (Meza et al ., 1980, f. Cell Biol ., 87 : 746-754.) . intermixed with "leaky" ones (7).As in the case of leaky epithelia, the monolayer has the ability to discriminate Na' from Cl -with a 9:1 permeability ratio, and the monovalent cations of the series IA follow Eisenman's 6th pattern (corresponding to negative sites with a medium force field) .The occluding junctions of MDCK monolayers can be opened by removing Ca" and adding EGTA to the bathing medium . Restoration of Ca" reseals the junction (5). functional resealing, which is completed in 2-3 h, offers the opportunity to study the factors that participate in the reassembly process of the cellular contacts by monitoring changes in transmural electrical resistance and by modifications of the freeze-fracture electron microscopy pattern of the junctions.

Permeability alterations of microvascular endothelia may be a factor in the plasma leakage produced by dengue virus infection. Confluent monolayers of the human dermal microvascular endothelial cell line HMEC-1 were utilized as an experimental model to study the cellular responses induced by the virus. Infected monolayers showed increased permeability for [ 3 H]mannitol, but no changes were observed for 4-70 kDa dextrans at 48 h post-infection (p.i.), a time at which viral titres reached maximal values and 40 % of the cells expressed viral proteins. A further increase in permeability occurred at 72 h, still without evident cytopathic effects on the monolayer. Coinciding with this, actin was reorganized in the infected cells and the tight junction protein occludin was displaced to the cytoplasm. Increments in the thickness of stress fibres and focal adhesions were observed in uninfected cells neighbouring infected cells. Culture medium from infected monolayers induced permeability changes and thickening of actin-containing structures in control cultures that resembled those observed 48 h p.i. Interleukin (IL) 8 was found in culture medium at concentrations ranging from 20 to 100 pg ml "1 . Neutralizing antibodies against IL8 partially inhibited the changes produced by the culture medium as well as those induced by addition of IL8. Genistein inhibited the effect of the culture medium and the phosphorylation of proteins associated with focal adhesions and indicated the participation of tyrosine kinases. These findings suggest that IL8 production by infected monolayers contributes to the virus-induced effect on the cytoskeleton and tight junctions and thereby modifies transendothelial permeability.

MDCK cells form monolayers which have many of the properties usually found in transporting epithelia. The present article is devoted to the study of the structural and functional polarization of MDCK cells, which is one of the central features of transporting epithelia. The results show: (i) that MDCK monolayers transport 2.6 mumol hr-1 cm-2 of sodium in the apical to basolateral direction; (ii) the passive flux of this ion is relatively large (20.3 mole hr-1 cm-2), which is a characteristic of leaky epithelia; (iii) a large fraction of the penetration of sodium into the cells proceeds through an amiloride-sensitive channel, and the exit is operated mainly by a ouabain-sensitive pump; (iv) the net transport of sodium from the apical to the basolateral side agrees with the asymmetric labeling of the pumps with 3H-ouabain; (v) this asymmetric labeling agrees, in turn, with a higher concentration of intramembrane particles (IMPs) in freeze-fracture replicas of the basolateral side of the plasma membrane; (vi) the structural polarization of confluent MDCK cells is also revealed by the location of microvilli, occluding junctions, and pinocytotic vesicles; and (vii) the presence of a continuous ring formed by actin microfilaments visualized by immunofluorescence under the lateral aspect of the plasma membrane that may be related to the distribution of the occluding junctions, which act as barriers separating apical from basolateral membrane components.

MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This article explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicine and cytochalasin B, on the degree of tightness of the occluding junctions . To study the degree of tightness, monolayers were prepared by plating MDCK cells on nylon disks coated with collagen . Disks were mounted as flat sheets between two Lucite chambers, and the'sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers . Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation . Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane . Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell . Colchicine causes a profound disruption of microtubules but only a 27% decrease in the electrical resistance of the resting monolayers . Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance . The cortical ring of filaments remains in place but appears fragmented with time . We find that removal of extracellular Ca", which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca". Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible . The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTAopened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.

When MDCK cells are cultured in monolayers, they synthesize, assemble, and seal occluding junctions that limit the paracellular route. These processes may be impaired by inhibitors of the protein synthesis but not by inhibitors of the synthesis of RNA. Once established, the occluding junctions confer to the monolayer an overall electrical resistance of 80-600 omega . cm2. At the microscopical level, the resistance of individual junctions have large variations along the perimeter of a given cell. This agrees with the images of freeze-fracture electron microscopy where the network of the junction varies abruptly from 1 to 10 strands. The junctions are impermeable to macromolecular tracers, have a 9 to 1 Na+/Cl- discrimination, and a cation selectivity following the order: K+ greater than Na+ greater than Rb+ greater than Cs+ greater than Li+. Sealing requires extracellular Ca2+, but the junctions open when the concentration of Ca2+ in the cytoplasm increases. The structural components of the cytoskeleton (microtubules and microfilaments) seem to be involved in the junctional events as revealed by staining with immunofluorescent specific antibodies. If the cells are treated with cytochalasin B, actin microfilaments disorganize, the junctions open, and the electrical resistance across the monolayers falls. The resealing of the tight junction is inhibited by this drug.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark ( Squalus acanthias) and the sea lamprey ( Petromyzon marinus) , exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

BackgroundMixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined.MethodologyTrophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified.Principal Findings E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica. ConclusionsEpithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage.SignificanceThe in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon.

Abstract. Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix.In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane poptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble ~25I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN.We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.