Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration.

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca(2+)-dependent microtubule disassembly and Ca(2+)-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.

Rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration.

Rotaviruses, which infect mature enterocytes of the small intestine, are recognized as the most important cause of viral gastroenteritis in young children. We have previously reported that rotavirus infection induces microvillar F-actin disassembly in human intestinal epithelial Caco-2 cells (N. Jourdan, J. P. Brunet, C. Sapin, A. Blais, J. Cotte-Laffitte, F. Forestier, A. M. Quero, G. Trugnan, and A. L. Servin, J. Virol. 72:7228-7236, 1998). In this study, to determine the mechanism responsible for rotavirus-induced F-actin alteration, we investigated the effect of infection on intracellular calcium concentration ([Ca(2+)](i)) in Caco-2 cells, since Ca(2+) is known to be a determinant factor for actin cytoskeleton regulation. As measured by quin2 fluorescence, viral replication induced a progressive increase in [Ca(2+)](i) from 7 h postinfection, which was shown to be necessary and sufficient for microvillar F-actin disassembly. During the first hours of infection, the increase in [Ca(2+)](i) was related only to an increase in Ca(2+) permeability of plasmalemma. At a late stage of infection, [Ca(2+)](i) elevation was due to both extracellular Ca(2+) influx and Ca(2+) release from the intracellular organelles, mainly the endoplasmic reticulum (ER). We noted that at this time the [Ca(2+)](i) increase was partially related to a phospholipase C (PLC)-dependent mechanism, which probably explains the Ca(2+) release from the ER. We also demonstrated for the first time that viral proteins or peptides, released into culture supernatants of rotavirus-infected Caco-2 cells, induced a transient increase in [Ca(2+)](i) of uninfected Caco-2 cells, by a PLC-dependent efflux of Ca(2+) from the ER and by extracellular Ca(2+) influx. These supernatants induced a Ca(2+)-dependent microvillar F-actin alteration in uninfected Caco-2 cells, thus participating in rotavirus pathogenesis.

Rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton.

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268-8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.

Three new bistetrahydrofuran acetogenins from the seeds of Annona spinescens.

Three new bistetrahydrofuran acetogenins, carolins A-C (1-3), were isolated from the MeOH extract of Annona spinescens in addition to the known compound, squamocin (4). The structures of 1, 2, and 3 were elucidated by spectroscopic methods including LSIMS/MS technique and confirmed by a chemical transformation, The cytotoxic activity of the new compounds 1-3 is reported and discussed in comparison with 4 and the previously isolated spinencin (5).

Infection of cultured human intestinal cells by monkey RRV and human Wa rotavirus as a function of intestinal epithelial cell differentiation.

Rotaviruses display in vivo a specific tropism for enterocytes of the small intestine. We examined here the infection of cultured human intestinal epithelial Caco-2 cells by rhesus monkey rotavirus (RRV) and human Wa rotavirus. The maximal infection of these cells was obtained when trypsin was present both in the viral inoculum before adsorption to the cells and in the culture medium during the course of cell infection. Since the differentiation process of Caco-2 cells in culture closely mimics in vivo differentiation of enterocytes along the crypt-villus axis, cell infection by RRV and Wa rotavirus was examined as a function of cell differentiation. We showed that RRV and Wa rotavirus can infect equally well both undifferentiated and differentiated Caco-2 cells.

Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus.

Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment.

Effect of acute oral administration of alcohol on superoxide anion production from mouse alveolar macrophages.

The production of superoxide anions (superoxide) from alveolar macrophages stimulated or not with opsonized zymosan was investigated in the mouse after acute oral administration of alcohol (6.5 g/kg). Superoxide production was assayed using a nitroblue tetrazolium (NBT) reduction and chemiluminescence assay. In the absence of opsonized zymosan, superoxide concentration was not affected 1 h after ethanol treatment but was significantly increased 15 and 24 h after treatment. In the presence of opsonized zymosan, a biphasic response was observed. Superoxide production was significantly reduced 1 and 3 h after administration but was increased 15 and 24 h after treatment. One hour after treatment, the percentage of cells that phagocytized opsonized zymosan and reduced NBT was significantly decreased, whereas 24 h after alcohol treatment, phagocytosis was normal and the percentage of cells reducing NBT was significantly increased. The activity of cytosolic superoxide dismutase from alveolar macrophages was not altered 1 h after administration but was significantly reduced 24 h later. Considering the functions of alveolar macrophages in the defense of the lung, these alterations in the production of reactive oxygen species after ingestion of alcohol could explain why alcoholics are more sensitive to pulmonary infections.

[Clostridium difficile and its cytotoxin in the stools of young hospitalized children. Influence of antibiotic treatment]. / Clostridium difficile et sa cytotoxine dans les selles de jeunes enfants hospitalisés. Influence du traitement antibiotique.

Clostridium difficile has been searched in 153 stool samples from 138 children aged 0 to 12 months. We divided the population in two groups depending on the antibiotic treatment. We have found C difficile in 39 samples (25%). The colonization rate increases with age ranging from 5% before 1 month, to 36% between 1 and 6 months and 54% between 6 and 13 months. An environmental sampling yielded once C difficile. Contamination may be related to the environment. 29% of the isolates produced a cytopathic toxin. Toxin titers in infants' stools range from 1/160 to 1/10240. One only of these children had diarrhea. C difficile and its toxin does not seem to infer any signs of enteric illness with infants. The results obtained with the group of non treated infants are not significantly different from the ones of the other group: the colonization rates are 21% in the non treated group and 29% in the other group. The rate of strains yielding a cytophatic toxin is similar in the 2 groups. It seems reasonable to agree that antibiotics do not influence the settlement of C difficile in infants' intestine.

[Augmentation by dilution of the effect of PVP iodine on poliomyelitis virus type I]. / Augmentation par la dilution de l'activité de la PVP iodée sur le virus poliomyelitique type I.

The influence of dilution of povidone-iodine solutions on virucidal activity against poliovirus type 1 was investigated. After exposure to different concentrations of povidone-iodine, the reduction in virus titer was determined after separating the antiseptic by gel filtration. Diluted preparations (0.5 and 0.25%) achieved a greater reduction in virus titer (10(5) in one hour) than the 5% stock solution (10(3)). Moreover, dilute solutions exhibited a faster virucidal activity.