Marisa Colone

Microenvironmental pH is a key factor for exosome traffic in tumor cells.

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model. Both mAbs bound to fungal cell surface and to the β1,3-β1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to β1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells. Our study demonstrates that the isotype of anti-β-glucan antibodies may affect details of the β-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

Immune Surveillance Properties of Human NK Cell-Derived Exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by “normal” cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Direct binding of human NK cell natural cytotoxicity receptor NKp44 to the surfaces of mycobacteria and other bacteria.

ABSTRACT Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56bright. In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

PE is a functional domain responsible for protein translocation and localization on mycobacterial cell wall.

The PE family of Mycobacterium tuberculosis includes 98 proteins which share a highly homologous N‐terminus sequence of about 110 amino acids (PE domain). Depending on the C‐terminal domain, the PE family can be divided in three subfamilies, the largest of which is the PE_PGRS with 61 members. In this study, we determined the cellular localization of three PE proteins by cell fractionation and immunoelectron microscopy by expressing chimeric epitope‐tagged recombinant proteins in Mycobacterium smegmatis. We demonstrate that the PE domain of PE_PGRS33 and PE11 (a protein constituted by the only PE domain) contains the information necessary for cell wall localization, and that they can be used as N‐terminal fusion partners to deliver a sufficiently long C‐terminus‐linked protein domain on the mycobacterial cell surface. Indeed, we demonstrate that PE_PGRS33 and Rv3097c (a lipase belonging to the PE family) are surface exposed and localize in the mycobacterial cell wall. Moreover, we found that PE_PGRS33 is easily extractable by detergents suggesting its localization in the mycobacterial outer membrane. Beyond defining the cellular localization of these proteins, and a function for their PE domains, these data open the interesting possibility to construct recombinant mycobacteria expressing heterologous antigens on their surface for vaccine purposes.

Effects of Mentha suaveolens Essential Oil Alone or in Combination with Other Drugs in Candida albicans

Candidosis is the most important cause of fungal infections in humans. The yeast Candida albicans can form biofilms, and it is known that microbial biofilms play an important role in human diseases and are very difficult to treat. The prolonged treatment with drugs has often resulted in failure and resistance. Due to the emergence of multidrug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to analyse the effects induced by essential oil of Mentha suaveolens Ehrh (EOMS) on Candida albicans and its potential synergism when used in combination with conventional drugs. Morphological differences between control and EOMS treated yeast cells or biofilms were observed by scanning electron microscopy and transmission electron microscopy (SEM and TEM resp.,). In order to reveal the presence of cell cycle alterations, flow cytometry analysis was carried out as well. The synergic action of EOMS was studied with the checkerboard method, and the cellular damage induced by different treatments was analysed by TEM. The results obtained have demonstrated both the effects of EOMS on C. albicans yeast cells and biofilms and the synergism of EOMS when used in combination with conventional antifungal drugs as fluconazole (FLC) and micafungin (MCFG), and therefore we can hypothesize on its potential use in therapy. Further studies are necessary to know its mechanism of action.

A NH2 tau fragment targets neuronal mitochondria at AD synapses: possible implications for neurodegeneration.

Synapses are ultrastructural sites for memory storage in brain, and synaptic damage is the best pathologic correlate of cognitive decline in Alzheimers disease (AD). Post-translational hyperphosphorylation, enzyme-mediated truncation, conformational modifications, and aggregation of tau protein into neurofibrillary tangles (NFTs) are hallmarks for a heterogeneous group of neurodegenerative disorders, so-called tauopathies. AD is a secondary tauopathy since it is pathologically distinguished by the presence of amyloid-beta (Abeta)-containing senile plaques and the presence of tau-positive NFTs in the neocortex and hippocampus. Here, we report that a 20-22 kDa NH2-truncated tau fragment is largely enriched in human mitochondria from cryopreserved synaptosomes of AD brains and that its amount in terminal fields correlates with the pathological synaptic changes and with the organelle functional impairment. This NH2-truncated tau form is also found in other human, not AD-tauopathies, while its presence in AD patients is linked to Abeta multimeric species and likely to pathology severity. Finally native, patient-derived, Abeta oligomers-enriched extracts likely impair the mitochondrial function by the in vitro production of 20-22 kDa NH2-tau fragments in mature human SY5Y and in rat hippocampal neurons. Thus our findings suggest that the mitochondrial NH2-derived tau peptide distribution may exacerbate the synapse degeneration occurring in tauopathies, including AD, and sustain the in vivo NH-2 tau cleavage inhibitors as an alternative drug discovery strategies for AD therapy.

Esculentin(1-21), an amphibian skin membrane-active peptide with potent activity on both planktonic and biofilm cells of the bacterial pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that forms sessile communities, named biofilms. The non-motile forms are very difficult to eradicate and are often associated with the establishment of persistent infections, especially in patients with cystic fibrosis. The resistance of P. aeruginosa to conventional antibiotics has become a growing health concern worldwide and has prompted the search for new anti-infective agents with new modes of action. Naturally occurring antimicrobial peptides (AMPs) represent promising future template candidates. Here we report on the potent activity and membrane-perturbing effects of the amphibian AMP esculentin(1-21), on both the free-living and sessile forms of P. aeruginosa, as a possible mechanism for biofilm disruption. Furthermore, the findings that esculentin(1-21) is able to prolong survival of animals in models of sepsis and pulmonary infection indicate that this peptide can be a promising template for the generation of new antibiotic formulations to advance care of infections caused by P. aeruginosa.

Src-Tyrosine kinases are major agents in mitochondrial tyrosine phosphorylation

Mitochondrial tyrosine phosphorylation is emerging as an important mechanism in regulating mitochondrial function. This article, aimed at identifying which kinases are the major agents in mitochondrial tyrosine phosphorylation, shows that this role should be attributed to Src family members. Indeed, various members of this family, for example, Fgr, Fyn, Lyn, c‐Src, are constitutively present in the internal structure of mitochondria as well as Csk, a key enzyme in the regulation of the activity of this family. By means of different approaches, biochemical fractioning, Western blotting and immunogold analysis “in situ” of phosphotyrosine signaling, evidence is reported on the existence of a signal transduction pathway from plasma membrane to mitochondria, resulting in increasing Src‐dependent mitochondrial tyrosine phosphorylation. The activation of Src kinases at mitochondrial level is associated with the proliferative status where several mitochondrial proteins are specifically tyrosine‐phosphorylated. J. Cell. Biochem. 104: 840–849, 2008.

Increase of Virulence and Its Phenotypic Traits in Drug-Resistant Strains of Candida albicans

ABSTRACT There is concern about the rise of antifungal drug resistance, but little is known about comparative biological properties and pathogenicity of drug-resistant strains. We generated fluconazole (FLC; CO23RFLC)- or micafungin (FK; CO23RFK)-resistant strains of Candida albicans by treating a FLC- and FK-susceptible strain of this fungus (CO23S) with stepwise-increasing concentrations of either drug. Molecular analyses showed that CO23RFLC had acquired markedly increased expression of the drug-resistance efflux pump encoded by the MDR1 gene, whereas CO23RFK had a homozygous mutation in the FSK1 gene. These genetic modifications did not alter to any extent the growth capacity of the drug-resistant strains in vitro, either at 28°C or at 37°C, but markedly increased their experimental pathogenicity in a systemic mouse infection model, as assessed by the overall mortality and target organ invasion. Interestingly, no apparent increase in the vaginopathic potential of the strains was observed with an estrogen-dependent rat vaginal infection. The increased pathogenicity of drug-resistant strains for systemic infection was associated with a number of biochemical and physiological changes, including (i) marked cellular alterations associated with a different expression and content of major cell wall polysaccharides, (ii) more rapid and extensive hypha formation in both liquid and solid media, and (iii) increased adherence to plastic and a propensity for biofilm formation. Overall, our data demonstrate that experimentally induced resistance to antifungal drugs, irrespective of drug family, can substantially divert C. albicans biology, affecting in particular biological properties of potential relevance for deep-seated candidiasis.