Fabiana Superti

Antirotaviral activity of milk proteins : lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29

Abstract Different milk proteins were analyzed for their inhibitory effect on either rotavirus-mediated agglutination of human erythrocytes or rotavirus infection of the human enterocyte-like cell line HT-29. Proteins investigated were α-lactalbumin, β-lactoglobulin, apo-lactoferrin, and Fe3+-lactoferrin, and their antiviral action was compared with the activity of mucin, a milk glycoprotein known to affect rotavirus infection. Results obtained demonstrated that β-lactoglobulin, apo- and Fe3+-lactoferrin are able to inhibit the replication of rotavirus in a dose-dependent manner, apo-lactoferrin being the most active. It was shown that apo-lactoferrin hinders virus attachment to cell receptors since it is able to bind the viral particles and to prevent both rotavirus haemagglutination and viral binding to susceptible cells. Moreover, this protein markedly inhibited rotavirus antigen synthesis and yield in HT-29 cells when added during the viral adsorption step or when it was present in the first hours of infection, suggesting that this protein interferes with the early phases of rotavirus infection.

Massive Secretion by T Cells Is Caused by HIV Nef in Infected Cells and by Nef Transfer to Bystander Cells

The HIV Nef protein mediates endocytosis of surface receptors that correlates with disease progression, but the link between this Nef function and HIV pathogenesis is not clear. Here, we report that Nef-mediated activation of membrane trafficking is bidirectional, connecting endocytosis with exocytosis as occurs in activated T cells. Nef expression induced an extensive secretory activity in infected and, surprisingly, also in noninfected T cells, leading to the massive release of microvesicle clusters, a phenotype observed in vitro and in 36%-87% of primary CD4 T cells from HIV-infected individuals. Consistent with exocytosis in noninfected cells, Nef is transferred to bystander cells upon cell-to-cell contact and subsequently induces secretion in an Erk1/2-dependent manner. Thus, HIV Nef alters membrane dynamics, mimicking those of activated T cells and causing a transfer of infected cell signaling (TOS) to bystander cells. This mechanism may help explain the detrimental effect on bystander cells seen in HIV infection.

Inhibition of poliovirus type 1 infection by iron-, manganese-and zinc-saturated lactoferrin

Abstract In this study we investigated the inhibitory activity of different milk proteins on poliovirus infection in Vero cells. Proteins analyzed were mucin, α-lactalbumin, β-lactoglobulin, and bovine and human lactoferrin. Viral cytopathic effect was not prevented by mucin, α-lactalbumin or β-lactoglobulin, whereas the lactoferrins tested were able to inhibit the replication of poliovirus in a dose-dependent manner. Further experiments were carried out in which apo- and native lactoferrin or lactoferrin fully saturated with ferric, manganese or zinc ions were added to the cells during different phases of viral infection. Results obtained demonstrated that all lactoferrins were able to prevent viral replication when present during the entire cycle of poliovirus infection or during the viral adsorption step. Only zinc lactoferrin strongly inhibited viral infection when incubated with the cells after the viral attachment, being the inhibition directly correlated with the degree of zinc saturation. Our results demonstrated that all lactoferrins interfered with an early phase of poliovirus infection; zinc lactoferrin was the sole compound capable of inhibiting a phase of infection subsequent to virus internalization into the host cells.

Antiviral Activity of Lactoferrin

In 1976, Matthews et al.19 reported the antiviral activity of milk proteins, underlining their possible clinical importance. Only recently, this effect has been ascribed mainly to lactoferrin (Lf). Lf was initially shown to exert a protective influence in mice infected with Friend leukemia virus15. Subsequently, a potent antiviral activity has been attributed to Lf, both in vitro towards cytomegalovirus (HCMV)9,7, herpes simplex virus (HSV)17, human immunodeficiency virus (HIV)7,29 and in vivo towards HSV-16 and HCMV25. Our groups provided evidence on the antiviral activity of Lf towards HSV-218, rotavirus28, and HIV22 infections (Table 1).

Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection

Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the anti-rotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86-258) and a small peptide (324-329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf.

Antiadenovirus activity of milk proteins: lactoferrin prevents viral infection

Different milk proteins were analysed for their inhibitory effect on adenovirus infection in vitro. Proteins investigated were mucin, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrin, and human lactoferrin. Results obtained demonstrated that mucin, alpha-lactalbumin, and beta-lactoglobulin did not prevent the viral cytopathic effect, whereas lactoferrin was able to inhibit adenovirus replication in a dose-dependent manner. Further experiments were carried out in which lactoferrin was added to the cells during different phases of viral infection. Results obtained showed that lactoferrin was able to prevent viral replication when added both before, or during the viral adsorption step, or when present during the entire replicative cycle of adenovirus, demonstrating that its action takes place on an early phase of viral replication.

Entry Pathway of Vesicular Stomatitis Virus into Different Host Cells

A biochemical and morphological investigation of the mechanism of entry of vesicular stomatitis virus (VSV) into host cells of mammalian (HeLa), avian (CER), piscine (EPC) and arthropod (Aedes albopictus) origin, is described. VSV was capable of infecting all cell lines tested by a endosome- and/or a lysosome-dependent step since ammonium chloride and amantadine blocked the early stages of infection. Complement-dependent immune lysis of infected host cells provided evidence that in none of the four different cell types examined did insertion of VSV antigens occur from the outside to any great extent on the cell surface. When the entry process was studied by electron microscopy, virus particles were seen to be bound to the cell surface at 0 degrees C. After warming at 37 degrees C for homeothermic cells or at 26 degrees C for poikilothermic cells, virus was detected within coated pits and coated vesicles and, later, in lysosomes. VSV entry was seen to take place by endocytosis in all four cell lines, which were derived from phylogenetically unrelated species.

Antiviral activity of lactoferrin towards naked viruses

It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.

Characterization of adherent-invasive Escherichia coli isolated from pediatric patients with inflammatory bowel disease†

Background: Crohns disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are characterized by an abnormal immunological response to commensal bacteria colonizing intestinal lumen and mucosa. Among the latter, strains of adherent‐invasive Escherichia coli (AIEC), capable of adhering to and invading epithelium, and to replicate in macrophages, have been described in CD adults. We aimed at identifying and characterizing AIEC strains in pediatric IBD. Methods: In all, 24 CD children, 10 UC, and 23 controls were investigated. Mucosal biopsies, taken during colonoscopy, were analyzed for the presence of AIEC strains by an adhesive‐invasive test. Protein expression of the specific AIEC receptor, the carcinoembryonic antigen‐related cell adhesion molecule 6 (CEACAM6), was evaluated by western blot and immunohistochemistry, while tumor necrosis factor alpha (TNF‐&agr;) and interleukin (IL)‐8 mRNA expression was detected by real‐time polymerase chain reaction (PCR), after bacterial infection. Transmission electron microscopy and trans‐epithelial electric resistance assays were performed on biopsies to assess bacteria‐induced morphological and functional epithelial alterations. Results: Two bacterial strains, EC15 and EC10, were found to adhere and invade the Caco2 cell line, similar to the well‐known AIEC strain LF82 (positive control): they upregulated CEACAM6, TNF‐&agr;, and IL‐8 gene/protein expression, in vitro and in cultured intestinal mucosa; they could also survive inside macrophages and damage the epithelial barrier integrity. Lesions in the inflamed tissues were associated with bacterial infection. Conclusions: This is the first study showing the presence of adhesive‐invasive bacteria strains in the inflamed tissues of children with IBD. Collective features of these strains indicate that they belong to the AIEC spectrum, suggesting their possible role in disease pathogenesis. (Inflamm Bowel Dis 2011)

Gangliosides as binding sites in SA-11 rotavirus infection of LLC-MK2 cells

The chemical nature of receptors involved in the attachment of simian rotavirus (SA-11) to a monkey kidney cell line (LLC-MK2) was investigated. Enzymic treatment of cells before virus infection indicated that membrane proteins and phospholipids are not involved in virus attachment, whereas sialic acid and galactose participate in the receptor structure to differing extents. Incubation of SA-11 with bovine brain gangliosides before infection strongly reduced its ability to bind to cell membranes. Similar experiments with individual purified gangliosides from bovine brain showed that virus infection was prevented by preincubation with GM1. Moreover, desialylated cells regained susceptibility to virus infection when coated with whole gangliosides or GM1 immediately after Clostridium perfringens neuraminidase treatment. The binding of SA-11 to whole gangliosides or GM1 was quantified by an ELISA procedure. The results suggest that gangliosides, mainly GM1, are part of the receptor structure for SA-11 of susceptible LLC-MK2 cells.