Biomedical subjects

Maria Grazia Ammendolia

Publications and source records attributed to Maria Grazia Ammendolia.

8 recordsLinked to original sources

PubMed2006

Bovine lactoferrin inhibits echovirus endocytic pathway by interacting with viral structural polypeptides.

Lactoferrin, an 80 kDa bi-globular iron-binding glycoprotein belonging to the transferrin family, is a pleiotropic factor with potent antimicrobial and immunomodulatory activities, present in breast milk, in mucosal secretions, and in the secondary granules of neutrophils. Recently, we have shown that bovine lactoferrin prevents the early phases of echovirus infection and also acts as a survival factor inhibiting viral-induced apoptosis. In the present research we investigated the mechanism of bovine lactoferrin anti-echoviral effect demonstrating that echovirus enters susceptible cells by an endocytic pathway and that lactoferrin treatment is able to prevent viral genome delivery into the cytoplasm. It is likely that lactoferrin interaction with echovirus capsid proteins induces alterations that stabilize the conformation of the virion making it resistant to uncoating. Taken together, the results of our study show that the inhibition of echovirus 6 infectivity by lactoferrin is dependent on its interaction not only with cell surface glycosaminoglycan chains but also with viral structural proteins demonstrating that this glycoprotein targets the virus entry process.

Ammonium Chloride↗

PubMed2005

Bovine lactoferrin peptidic fragments involved in inhibition of Echovirus 6 in vitro infection.

Bovine lactoferrin is a multifunctional glycoprotein folded in two symmetric globular lobes (N- and C-lobes), each being able to bind one ferric ion. We have previously demonstrated that this protein is able to prevent echovirus-induced apoptosis. In the present study, we have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin effect on echovirus infection. Results obtained showed that bovine lactoferrin inhibits echovirus-induced cytopathic effect and antigen synthesis in a dose-dependent manner and that this protein is able to prevent viral replication when added not only during the entire cycle of infection but also before, during or after the viral adsorption step. The N-terminal cationic peptide was sufficient to prevent viral binding. Our data suggest that lactoferrin inhibition of echovirus attachment to cell receptors could be mediated by the cluster of positive charges at its N-terminus (lactoferricin).

Animals↗

PubMed2005

Glycosaminoglycans mediate invasion and survival of Enterococcus faecalis into macrophages.

Enterococcus faecalis is responsible for a large variety of nosocomial infections. The intestinal barrier is thought to be one of the preferential portals of entry of enterococci, and the ability of E. faecalis to survive within peritoneal macrophages may contribute to spreading to distant sites. We examined the ability of a polysaccharide-expressing (biofilm-positive) E. faecalis strain and an isogenic biofilm-negative mutant to enter and survive within professional and nonprofessional phagocytes. Biofilm-positive bacteria survived longer in all cell systems than did biofilm-negative bacteria, through a process of receptor-mediated endocytosis that is dependent on functional reorganization of microtubules and polymerization of microfilament and on activation of protein kinases but not ATPases or protein phosphatases. We suggest that glycosaminoglycans--specifically heparin, heparan sulfate, and chondroitin sulfate A--are the host receptors for enterococci on professional and, possibly, nonprofessional phagocytes, allowing entry of enterococci into cell compartments where killing mechanisms are inhibited.

Animals↗

PubMed2005

Inhibitory activity of bovine lactoferrin against echovirus induced programmed cell death in vitro.

Lactoferrin is a glycoprotein and plays an important role in defence against pathogens. Although the antiviral activity of lactoferrin is one of the major biological functions of such protein, the mechanism of action is still under debate. The effect of lactoferrin on echovirus 6 infection in vitro was analysed and results showed that (i) cells infected with echovirus 6, died as a result of apoptosis and that (ii) programmed cell death was inhibited by lactoferrin treatment. In this report, we demonstrate that lactoferrin can exert its anti-enteroviral activity by preventing viral-induced apoptosis.

Animals↗

PubMed2004

A Sphingomonas bacterium interacting with epithelial cells.

Bacteria of the genus Sphingomonas are environmental organisms that have recently been implicated in a variety of community-acquired and nosocomial infections. During studies on bacteria-cell interactions, we incurred a microorganism contaminating our HeLa cell culture, possibly from water utilized for reagent preparation; this bacterium appeared to tightly adhere to cell monolayers and to survive, with only limited growth rate, which did not seem to alter cells as far as shape, growth rate or survival were concerned. The contaminating organism was isolated and partially characterized by morphological, genetic, and biochemical assays. Mechanisms of cell interaction and entry into epithelial cells were investigated by electron microscopy, immunofluorescence, and biochemical inhibitors. Morphological and biochemical features indicated that the microorganism belonged to the genus Sphingomonas. Electron microscopy showed that contact between the Sphingomonas bacterium and epithelial cells leads to a dramatic alteration of the cell surface, with formation of numerous microvillar extensions plus membrane ruffling. Confocal microscopy and the use of inhibitors showed that actin microfilaments were involved during attachment and entry into HeLa cells. Macropinosome formation and an inhibitory effect by amiloride indicate that internalization occurs in part via a macropinocytosis mechanism. Moreover, cholesterol distribution at the site of bacterial binding suggests that Sphingomonas bacteria could use the lipid rafts as initial binding sites.

Actins↗

PubMed2004

Receptor-mediated endocytosis of biofilm-forming Enterococcus faecalis by rat peritoneal macrophages.

BACKGROUND & OBJECTIVES: Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics. Studies were taken up to identify virulence factors and to characterise pathogenic mechanisms of such infections to evaluate potential targets for treatments alternative to antibiotic therapy. This study was carried out to evaluate the contribution of extracellular polysaccharide expressed by Enterococcus faecalis to resistance to phagocytosis and survival within rat peritoneal macrophages. METHODS: Six E. faecalis clinical isolates were tested for their ability to survive within rat peritoneal macrophages. Cytochalasin D, colchicine and monodansylcadaverine were used to investigate the route of enterococcal entry inside macrophages. RESULTS: Four of the isolates were able to produce extracellular polysaccharide and form biofilm after growth in glucose-supplemented medium, while no production could be detected in glucose deficient medium. Two isolates were polysaccharide-negative in both conditions. Isolates expressing extracellular polysaccharide were able to survive for more than 24 h compared to polysaccharide-negative bacterial cells of the same strain grown in glucose-deficient medium, which were readily cleared. Cytochalasin D virtually abolished the number of viable intracellular bacteria, after growth in either trypticase soy broth (TSB) or TSB supplemented with glucose; colchicine and monodansylcadaverine strongly affected survival of polysaccharide-positive bacteria, significantly more than that of polysaccharide-negative ones. INTERPRETATION & CONCLUSION: Biofilm-forming E. faecalis survived within rat peritoneal macrophages significantly better than polysaccharide-negative isolates. Perturbators of cytoskeleton and of surface receptors turnover, indicated receptors-mediated endocytosis as the most likely route for enterococcal entry into macrophages.

Animals↗

PubMed2002

Invasion of HeLa cells by Enterococcus faecalis clinical isolates.

We examined the in vitro ability of Enterococcus faecalis clinical isolates to adhere to and to invade HeLa cells, suggested to be a valuable model system to study bacteria-directed endocytosis. Using a variety of compounds that act on eukaryotic cell structures, both microtubules and microfilaments were found to be involved in enterococcal entry into cells. Two distinct modes of interaction were observed: in one, a close proximity of bacteria with the cell membrane was observed, possibly leading to direct engulfment of the bacterial cell. In the other mode, cellular pseudopodal formation seemed to be stimulated by vicinity of bacterial cells; in some cases, such associations involved formation of clathrin-coated-like vesicles before internalizing enterococci. The above-mentioned experimental data together with the use of monodansylcadaverine, amiloride and NH4Cl, all involved in cytosol acidification and inhibition of receptor-mediated endocytosis (RME), led us to conclude that E. faecalis is internalized within HeLa cells by more than one invasion pathway. One, sensitive to amiloride, is most likely a macropinocytic, actin-dependent uptake mechanism, which determines the production of large smooth-membrane vacuoles engulfing enterococci. The other is RME, in which entry is dependent on both microfilament and microtubule structural integrity.

Acids↗

PubMed2000

Virulence and drug susceptibility of Mycobacterium celatum.

The virulence and drug susceptibility of a clinical isolate of Mycobacterium celatum which showed smooth transparent (ST) and smooth opaque (SO) colonies were studied. While ST cells multiplied intracellularly and maintained their coccobacillary form in a human macrophage model of infection, SO cells formed long filaments and completely destroyed the phagocytes. In BALB/c mice, the ST variant, but not the SO variant, grew efficiently in the spleen, liver and lung. The ST variant was usually more resistant in vitro than the SO variant to drugs, with MIC values for clarithromycin (CLA), azithromycin (AZI), ciprofloxacin, sparfloxacin, amikacin, clofazimine, ethambutol and isoniazid being higher than those of the SO variant. In beige mice infected with the more highly virulent variant ST, CLA and AZI were the most active drugs in terms of viable count reduction in organs and mutant selection. Together, these observations indicate that the ST variant of M. celatum is a virulent form that can be efficiently inhibited in vivo by CLA and AZI.

Animals↗